Structured Review

Millipore rnase a
Labeling profile of the targeted residues in <t>RNase</t> A on glass, PMMA, and HDPE surfaces when adsorbed from 1.00 mg/mL protein solutions. The residues within the active site of RNase A are shown separately in the right-hand plot to more clearly show their response. Profiles within about ±0.1 of zero are not significantly different from those in the solution state ( n = 3). Residues showing no difference in their solvation between the solution and adsorbed states have profile values equal to 0 (e.g., R85 for the glass and HDPE surfaces).
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Images

1) Product Images from "Adsorption-Induced Changes in Ribonuclease A Structure and Enzymatic Activity on Solid Surfaces"

Article Title: Adsorption-Induced Changes in Ribonuclease A Structure and Enzymatic Activity on Solid Surfaces

Journal: Langmuir

doi: 10.1021/la503854a

Labeling profile of the targeted residues in RNase A on glass, PMMA, and HDPE surfaces when adsorbed from 1.00 mg/mL protein solutions. The residues within the active site of RNase A are shown separately in the right-hand plot to more clearly show their response. Profiles within about ±0.1 of zero are not significantly different from those in the solution state ( n = 3). Residues showing no difference in their solvation between the solution and adsorbed states have profile values equal to 0 (e.g., R85 for the glass and HDPE surfaces).
Figure Legend Snippet: Labeling profile of the targeted residues in RNase A on glass, PMMA, and HDPE surfaces when adsorbed from 1.00 mg/mL protein solutions. The residues within the active site of RNase A are shown separately in the right-hand plot to more clearly show their response. Profiles within about ±0.1 of zero are not significantly different from those in the solution state ( n = 3). Residues showing no difference in their solvation between the solution and adsorbed states have profile values equal to 0 (e.g., R85 for the glass and HDPE surfaces).

Techniques Used: Labeling

Space-filled model of RNase A with amino acid residues color coded by their solvent accessibility, as determined from targeted amino acid labeling in solution. Color coding: charged amino acid residues (Asp, Glu, Lys, Arg, His) with high solvent accessibility (green) and moderate solvent accessibility (blue), tyrosine residues with high solvent accessibility (orange) and low solvent accessibility (black). Nontargeted amino acid residues are color coded in light gray. Figure illustrated using UCSF Chimera. The arrows point out the location of the three key amino acid residues that provide the catalytic function of the enzyme (H12, K41, H119).
Figure Legend Snippet: Space-filled model of RNase A with amino acid residues color coded by their solvent accessibility, as determined from targeted amino acid labeling in solution. Color coding: charged amino acid residues (Asp, Glu, Lys, Arg, His) with high solvent accessibility (green) and moderate solvent accessibility (blue), tyrosine residues with high solvent accessibility (orange) and low solvent accessibility (black). Nontargeted amino acid residues are color coded in light gray. Figure illustrated using UCSF Chimera. The arrows point out the location of the three key amino acid residues that provide the catalytic function of the enzyme (H12, K41, H119).

Techniques Used: Labeling

Labeling profile of the targeted residues in RNase A on glass, PMMA, and HDPE surfaces when adsorbed from 0.03 mg/mL protein solution. The residues within the active site of RNase A are shown separately in the right-hand plot to more clearly show their response. The profiles within about ±0.1 of zero are not significantly different from those in the solution state ( n = 3). Residues showing no difference in their solvation between the solution and adsorbed states have profile values equal to 0 (e.g., Y115 for all three surfaces).
Figure Legend Snippet: Labeling profile of the targeted residues in RNase A on glass, PMMA, and HDPE surfaces when adsorbed from 0.03 mg/mL protein solution. The residues within the active site of RNase A are shown separately in the right-hand plot to more clearly show their response. The profiles within about ±0.1 of zero are not significantly different from those in the solution state ( n = 3). Residues showing no difference in their solvation between the solution and adsorbed states have profile values equal to 0 (e.g., Y115 for all three surfaces).

Techniques Used: Labeling

Solvation profiles of residues in RNase A adsorbed in (A) 0.03 and (B) 1.00 mg/mL on the PMMA surface. Residue color code: yellow (− −), orange (−), dark gray (native state), green (+), blue (++), and light gray (nontargeted). The arrows point to the location of the three key amino acid residues that provide the catalytic function of the enzyme (H12, K41, and H119).
Figure Legend Snippet: Solvation profiles of residues in RNase A adsorbed in (A) 0.03 and (B) 1.00 mg/mL on the PMMA surface. Residue color code: yellow (− −), orange (−), dark gray (native state), green (+), blue (++), and light gray (nontargeted). The arrows point to the location of the three key amino acid residues that provide the catalytic function of the enzyme (H12, K41, and H119).

Techniques Used:

Solvation profile of residues in RNase A adsorbed in (A) 0.03 and (B) 1.00 mg/mL on the HDPE surface. Residue color code: yellow (− −), orange (−), dark gray (native state), green (+), blue (++), and light gray (nontargeted). The arrows point to the location of the three key amino acid residues that provide the catalytic function of the enzyme (H12, K41, and H119).
Figure Legend Snippet: Solvation profile of residues in RNase A adsorbed in (A) 0.03 and (B) 1.00 mg/mL on the HDPE surface. Residue color code: yellow (− −), orange (−), dark gray (native state), green (+), blue (++), and light gray (nontargeted). The arrows point to the location of the three key amino acid residues that provide the catalytic function of the enzyme (H12, K41, and H119).

Techniques Used:

Ribbon diagram of the three-dimensional structure of ribonuclease A.  45  The three residues most important for catalysis, His12, His119, and Lys41, are marked in red.
Figure Legend Snippet: Ribbon diagram of the three-dimensional structure of ribonuclease A. 45 The three residues most important for catalysis, His12, His119, and Lys41, are marked in red.

Techniques Used:

Solvation profile of residues in RNase A adsorbed from (A) 0.03 and (B) 1.00 mg/mL on the glass surface. Residue color code: yellow (− −), orange (−), dark gray (native state), green (+), blue (++), and light gray (nontargeted). The arrows point to the location of the three key amino acid residues that provide the catalytic function of the enzyme (H12, K41, and H119).
Figure Legend Snippet: Solvation profile of residues in RNase A adsorbed from (A) 0.03 and (B) 1.00 mg/mL on the glass surface. Residue color code: yellow (− −), orange (−), dark gray (native state), green (+), blue (++), and light gray (nontargeted). The arrows point to the location of the three key amino acid residues that provide the catalytic function of the enzyme (H12, K41, and H119).

Techniques Used:

2) Product Images from "Adsorption-Induced Changes in Ribonuclease A Structure and Enzymatic Activity on Solid Surfaces"

Article Title: Adsorption-Induced Changes in Ribonuclease A Structure and Enzymatic Activity on Solid Surfaces

Journal: Langmuir

doi: 10.1021/la503854a

Labeling profile of the targeted residues in RNase A on glass, PMMA, and HDPE surfaces when adsorbed from 1.00 mg/mL protein solutions. The residues within the active site of RNase A are shown separately in the right-hand plot to more clearly show their response. Profiles within about ±0.1 of zero are not significantly different from those in the solution state ( n = 3). Residues showing no difference in their solvation between the solution and adsorbed states have profile values equal to 0 (e.g., R85 for the glass and HDPE surfaces).
Figure Legend Snippet: Labeling profile of the targeted residues in RNase A on glass, PMMA, and HDPE surfaces when adsorbed from 1.00 mg/mL protein solutions. The residues within the active site of RNase A are shown separately in the right-hand plot to more clearly show their response. Profiles within about ±0.1 of zero are not significantly different from those in the solution state ( n = 3). Residues showing no difference in their solvation between the solution and adsorbed states have profile values equal to 0 (e.g., R85 for the glass and HDPE surfaces).

Techniques Used: Labeling

Space-filled model of RNase A with amino acid residues color coded by their solvent accessibility, as determined from targeted amino acid labeling in solution. Color coding: charged amino acid residues (Asp, Glu, Lys, Arg, His) with high solvent accessibility (green) and moderate solvent accessibility (blue), tyrosine residues with high solvent accessibility (orange) and low solvent accessibility (black). Nontargeted amino acid residues are color coded in light gray. Figure illustrated using UCSF Chimera. The arrows point out the location of the three key amino acid residues that provide the catalytic function of the enzyme (H12, K41, H119).
Figure Legend Snippet: Space-filled model of RNase A with amino acid residues color coded by their solvent accessibility, as determined from targeted amino acid labeling in solution. Color coding: charged amino acid residues (Asp, Glu, Lys, Arg, His) with high solvent accessibility (green) and moderate solvent accessibility (blue), tyrosine residues with high solvent accessibility (orange) and low solvent accessibility (black). Nontargeted amino acid residues are color coded in light gray. Figure illustrated using UCSF Chimera. The arrows point out the location of the three key amino acid residues that provide the catalytic function of the enzyme (H12, K41, H119).

Techniques Used: Labeling

Labeling profile of the targeted residues in RNase A on glass, PMMA, and HDPE surfaces when adsorbed from 0.03 mg/mL protein solution. The residues within the active site of RNase A are shown separately in the right-hand plot to more clearly show their response. The profiles within about ±0.1 of zero are not significantly different from those in the solution state ( n = 3). Residues showing no difference in their solvation between the solution and adsorbed states have profile values equal to 0 (e.g., Y115 for all three surfaces).
Figure Legend Snippet: Labeling profile of the targeted residues in RNase A on glass, PMMA, and HDPE surfaces when adsorbed from 0.03 mg/mL protein solution. The residues within the active site of RNase A are shown separately in the right-hand plot to more clearly show their response. The profiles within about ±0.1 of zero are not significantly different from those in the solution state ( n = 3). Residues showing no difference in their solvation between the solution and adsorbed states have profile values equal to 0 (e.g., Y115 for all three surfaces).

Techniques Used: Labeling

Solvation profiles of residues in RNase A adsorbed in (A) 0.03 and (B) 1.00 mg/mL on the PMMA surface. Residue color code: yellow (− −), orange (−), dark gray (native state), green (+), blue (++), and light gray (nontargeted). The arrows point to the location of the three key amino acid residues that provide the catalytic function of the enzyme (H12, K41, and H119).
Figure Legend Snippet: Solvation profiles of residues in RNase A adsorbed in (A) 0.03 and (B) 1.00 mg/mL on the PMMA surface. Residue color code: yellow (− −), orange (−), dark gray (native state), green (+), blue (++), and light gray (nontargeted). The arrows point to the location of the three key amino acid residues that provide the catalytic function of the enzyme (H12, K41, and H119).

Techniques Used:

Solvation profile of residues in RNase A adsorbed in (A) 0.03 and (B) 1.00 mg/mL on the HDPE surface. Residue color code: yellow (− −), orange (−), dark gray (native state), green (+), blue (++), and light gray (nontargeted). The arrows point to the location of the three key amino acid residues that provide the catalytic function of the enzyme (H12, K41, and H119).
Figure Legend Snippet: Solvation profile of residues in RNase A adsorbed in (A) 0.03 and (B) 1.00 mg/mL on the HDPE surface. Residue color code: yellow (− −), orange (−), dark gray (native state), green (+), blue (++), and light gray (nontargeted). The arrows point to the location of the three key amino acid residues that provide the catalytic function of the enzyme (H12, K41, and H119).

Techniques Used:

Ribbon diagram of the three-dimensional structure of ribonuclease A.  45  The three residues most important for catalysis, His12, His119, and Lys41, are marked in red.
Figure Legend Snippet: Ribbon diagram of the three-dimensional structure of ribonuclease A. 45 The three residues most important for catalysis, His12, His119, and Lys41, are marked in red.

Techniques Used:

Solvation profile of residues in RNase A adsorbed from (A) 0.03 and (B) 1.00 mg/mL on the glass surface. Residue color code: yellow (− −), orange (−), dark gray (native state), green (+), blue (++), and light gray (nontargeted). The arrows point to the location of the three key amino acid residues that provide the catalytic function of the enzyme (H12, K41, and H119).
Figure Legend Snippet: Solvation profile of residues in RNase A adsorbed from (A) 0.03 and (B) 1.00 mg/mL on the glass surface. Residue color code: yellow (− −), orange (−), dark gray (native state), green (+), blue (++), and light gray (nontargeted). The arrows point to the location of the three key amino acid residues that provide the catalytic function of the enzyme (H12, K41, and H119).

Techniques Used:

3) Product Images from "Adsorption-Induced Changes in Ribonuclease A Structure and Enzymatic Activity on Solid Surfaces"

Article Title: Adsorption-Induced Changes in Ribonuclease A Structure and Enzymatic Activity on Solid Surfaces

Journal: Langmuir

doi: 10.1021/la503854a

Labeling profile of the targeted residues in RNase A on glass, PMMA, and HDPE surfaces when adsorbed from 1.00 mg/mL protein solutions. The residues within the active site of RNase A are shown separately in the right-hand plot to more clearly show their response. Profiles within about ±0.1 of zero are not significantly different from those in the solution state ( n = 3). Residues showing no difference in their solvation between the solution and adsorbed states have profile values equal to 0 (e.g., R85 for the glass and HDPE surfaces).
Figure Legend Snippet: Labeling profile of the targeted residues in RNase A on glass, PMMA, and HDPE surfaces when adsorbed from 1.00 mg/mL protein solutions. The residues within the active site of RNase A are shown separately in the right-hand plot to more clearly show their response. Profiles within about ±0.1 of zero are not significantly different from those in the solution state ( n = 3). Residues showing no difference in their solvation between the solution and adsorbed states have profile values equal to 0 (e.g., R85 for the glass and HDPE surfaces).

Techniques Used: Labeling

Space-filled model of RNase A with amino acid residues color coded by their solvent accessibility, as determined from targeted amino acid labeling in solution. Color coding: charged amino acid residues (Asp, Glu, Lys, Arg, His) with high solvent accessibility (green) and moderate solvent accessibility (blue), tyrosine residues with high solvent accessibility (orange) and low solvent accessibility (black). Nontargeted amino acid residues are color coded in light gray. Figure illustrated using UCSF Chimera. The arrows point out the location of the three key amino acid residues that provide the catalytic function of the enzyme (H12, K41, H119).
Figure Legend Snippet: Space-filled model of RNase A with amino acid residues color coded by their solvent accessibility, as determined from targeted amino acid labeling in solution. Color coding: charged amino acid residues (Asp, Glu, Lys, Arg, His) with high solvent accessibility (green) and moderate solvent accessibility (blue), tyrosine residues with high solvent accessibility (orange) and low solvent accessibility (black). Nontargeted amino acid residues are color coded in light gray. Figure illustrated using UCSF Chimera. The arrows point out the location of the three key amino acid residues that provide the catalytic function of the enzyme (H12, K41, H119).

Techniques Used: Labeling

Labeling profile of the targeted residues in RNase A on glass, PMMA, and HDPE surfaces when adsorbed from 0.03 mg/mL protein solution. The residues within the active site of RNase A are shown separately in the right-hand plot to more clearly show their response. The profiles within about ±0.1 of zero are not significantly different from those in the solution state ( n = 3). Residues showing no difference in their solvation between the solution and adsorbed states have profile values equal to 0 (e.g., Y115 for all three surfaces).
Figure Legend Snippet: Labeling profile of the targeted residues in RNase A on glass, PMMA, and HDPE surfaces when adsorbed from 0.03 mg/mL protein solution. The residues within the active site of RNase A are shown separately in the right-hand plot to more clearly show their response. The profiles within about ±0.1 of zero are not significantly different from those in the solution state ( n = 3). Residues showing no difference in their solvation between the solution and adsorbed states have profile values equal to 0 (e.g., Y115 for all three surfaces).

Techniques Used: Labeling

Solvation profiles of residues in RNase A adsorbed in (A) 0.03 and (B) 1.00 mg/mL on the PMMA surface. Residue color code: yellow (− −), orange (−), dark gray (native state), green (+), blue (++), and light gray (nontargeted). The arrows point to the location of the three key amino acid residues that provide the catalytic function of the enzyme (H12, K41, and H119).
Figure Legend Snippet: Solvation profiles of residues in RNase A adsorbed in (A) 0.03 and (B) 1.00 mg/mL on the PMMA surface. Residue color code: yellow (− −), orange (−), dark gray (native state), green (+), blue (++), and light gray (nontargeted). The arrows point to the location of the three key amino acid residues that provide the catalytic function of the enzyme (H12, K41, and H119).

Techniques Used:

Solvation profile of residues in RNase A adsorbed in (A) 0.03 and (B) 1.00 mg/mL on the HDPE surface. Residue color code: yellow (− −), orange (−), dark gray (native state), green (+), blue (++), and light gray (nontargeted). The arrows point to the location of the three key amino acid residues that provide the catalytic function of the enzyme (H12, K41, and H119).
Figure Legend Snippet: Solvation profile of residues in RNase A adsorbed in (A) 0.03 and (B) 1.00 mg/mL on the HDPE surface. Residue color code: yellow (− −), orange (−), dark gray (native state), green (+), blue (++), and light gray (nontargeted). The arrows point to the location of the three key amino acid residues that provide the catalytic function of the enzyme (H12, K41, and H119).

Techniques Used:

Ribbon diagram of the three-dimensional structure of ribonuclease A.  45  The three residues most important for catalysis, His12, His119, and Lys41, are marked in red.
Figure Legend Snippet: Ribbon diagram of the three-dimensional structure of ribonuclease A. 45 The three residues most important for catalysis, His12, His119, and Lys41, are marked in red.

Techniques Used:

Solvation profile of residues in RNase A adsorbed from (A) 0.03 and (B) 1.00 mg/mL on the glass surface. Residue color code: yellow (− −), orange (−), dark gray (native state), green (+), blue (++), and light gray (nontargeted). The arrows point to the location of the three key amino acid residues that provide the catalytic function of the enzyme (H12, K41, and H119).
Figure Legend Snippet: Solvation profile of residues in RNase A adsorbed from (A) 0.03 and (B) 1.00 mg/mL on the glass surface. Residue color code: yellow (− −), orange (−), dark gray (native state), green (+), blue (++), and light gray (nontargeted). The arrows point to the location of the three key amino acid residues that provide the catalytic function of the enzyme (H12, K41, and H119).

Techniques Used:

4) Product Images from "Modeling formalin fixation and antigen retrieval with bovine pancreatic ribonuclease A: I--Structural and functional alterations"

Article Title: Modeling formalin fixation and antigen retrieval with bovine pancreatic ribonuclease A: I--Structural and functional alterations

Journal:

doi: 10.1038/labinvest.3700045

SDS–PAGE of formaldehyde-treated RNase A fractions taken before (lanes 1–3) and recovered after (lanes 4–6) DSC. Lanes: 1 and 4, monomer; 2 and 5, dimer; 3 and 6, mixture of oligomers higher than pentamers; M, molecular mass markers
Figure Legend Snippet: SDS–PAGE of formaldehyde-treated RNase A fractions taken before (lanes 1–3) and recovered after (lanes 4–6) DSC. Lanes: 1 and 4, monomer; 2 and 5, dimer; 3 and 6, mixture of oligomers higher than pentamers; M, molecular mass markers

Techniques Used: SDS Page

Near-UV CD spectra of native RNase A (profile 1) and RNase A (6.5 mg/ml) kept in 10% buffered formalin (pH 7.4) at 23°C for 210.5 h (profile 2), and their difference spectrum (profile 3). Spectrum 2 was recorded from a 10-fold diluted aliquot
Figure Legend Snippet: Near-UV CD spectra of native RNase A (profile 1) and RNase A (6.5 mg/ml) kept in 10% buffered formalin (pH 7.4) at 23°C for 210.5 h (profile 2), and their difference spectrum (profile 3). Spectrum 2 was recorded from a 10-fold diluted aliquot

Techniques Used:

Far-UV CD spectra of native RNase A (profile 1) and RNase A (6.5 mg/ml) kept in 10% buffered formalin (pH 7.4) at 23°C for 210.4 h (profile 2), and their difference spectrum (profile 3). Spectrum 2 was recorded from the undiluted reaction mixture.
Figure Legend Snippet: Far-UV CD spectra of native RNase A (profile 1) and RNase A (6.5 mg/ml) kept in 10% buffered formalin (pH 7.4) at 23°C for 210.4 h (profile 2), and their difference spectrum (profile 3). Spectrum 2 was recorded from the undiluted reaction mixture.

Techniques Used:

Time course of the activity restoration of the formaldehyde-treated RNase A during incubation at 50°C (0–2 h) and 65°C (2–4 h) in TAE buffer (pH 7.0).
Figure Legend Snippet: Time course of the activity restoration of the formaldehyde-treated RNase A during incubation at 50°C (0–2 h) and 65°C (2–4 h) in TAE buffer (pH 7.0).

Techniques Used: Activity Assay, Incubation

Heat absorption in solutions of native RNase A (profile 1) and RNase A kept in 10% buffered formalin for 47.5 (profile 2) and 138.8 h (profile 3) at pH 7.4 and 23°C. All samples were dialyzed against 75 mM potassium phosphate buffer (pH 7.4) prior
Figure Legend Snippet: Heat absorption in solutions of native RNase A (profile 1) and RNase A kept in 10% buffered formalin for 47.5 (profile 2) and 138.8 h (profile 3) at pH 7.4 and 23°C. All samples were dialyzed against 75 mM potassium phosphate buffer (pH 7.4) prior

Techniques Used:

Dependence of RNase A T d of the dialyzed samples on time of incubation in 10% buffered formalin at pH 7.4 and 23°C.
Figure Legend Snippet: Dependence of RNase A T d of the dialyzed samples on time of incubation in 10% buffered formalin at pH 7.4 and 23°C.

Techniques Used: Incubation

Heat absorption of solutions of formaldehyde-treated RNase A chromatography fractions: 1, monomer; 2, dimer; 3, mixture of oligomers higher than pentamers. The protein concentration was 0.55 (profile 1), 0.36 (profile 2), or 0.37 mg/ml (profile 3).
Figure Legend Snippet: Heat absorption of solutions of formaldehyde-treated RNase A chromatography fractions: 1, monomer; 2, dimer; 3, mixture of oligomers higher than pentamers. The protein concentration was 0.55 (profile 1), 0.36 (profile 2), or 0.37 mg/ml (profile 3).

Techniques Used: Chromatography, Protein Concentration

IEF of formaldehyde-treated RNase A (A) and its fractions (B) separated by gel filtration. ( a ) Lanes: M, IEF markers; 1, unfractionated formaldehyde-treated RNase A. ( b ) Lanes: M, IEF markers; 1, monomer; 2, dimer; 3, trimer; 4, tetramer; 5, pentamer.
Figure Legend Snippet: IEF of formaldehyde-treated RNase A (A) and its fractions (B) separated by gel filtration. ( a ) Lanes: M, IEF markers; 1, unfractionated formaldehyde-treated RNase A. ( b ) Lanes: M, IEF markers; 1, monomer; 2, dimer; 3, trimer; 4, tetramer; 5, pentamer.

Techniques Used: Electrofocusing, Filtration

( a ) SDS–PAGE of RNase A at 6.5 mg/ml before (lane 1) and after incubation in 10% buffered formalin for 0.3 (lane 2), 23.4 (lane 3), 47.5 (lane 4), 71.9 (lane 5), 138.8 (lane 6), and 210.5 (lane 7) h at 23°C. ( b ) SDS–PAGE of native
Figure Legend Snippet: ( a ) SDS–PAGE of RNase A at 6.5 mg/ml before (lane 1) and after incubation in 10% buffered formalin for 0.3 (lane 2), 23.4 (lane 3), 47.5 (lane 4), 71.9 (lane 5), 138.8 (lane 6), and 210.5 (lane 7) h at 23°C. ( b ) SDS–PAGE of native

Techniques Used: SDS Page, Incubation

Time course of the activity restoration of the formaldehyde-treated RNase A during incubation at 65°C in TAE buffers of various pH values.
Figure Legend Snippet: Time course of the activity restoration of the formaldehyde-treated RNase A during incubation at 65°C in TAE buffers of various pH values.

Techniques Used: Activity Assay, Incubation

5) Product Images from "MicroRNA Drop in the Bloodstream and MicroRNA Boost in the Tumour Caused by Treatment with Ribonuclease A Leads to an Attenuation of Tumour Malignancy"

Article Title: MicroRNA Drop in the Bloodstream and MicroRNA Boost in the Tumour Caused by Treatment with Ribonuclease A Leads to an Attenuation of Tumour Malignancy

Journal: PLoS ONE

doi: 10.1371/journal.pone.0083482

Hypothetical mechanism of the effects of RNase A on the biogenesis of tumour-derived and circulating miRNAs. A . In tumour cells, positively charged RNase A binds with negatively charged tumour tissue (due to the presence of anion phosphatidylserine residues on the surface of tumour blood vessels) and penetrate into the cells. RNase A-mediated increase in miRNA levels in tumour cells may be a result of the activity of RNase A or its proteolytic fragment as a transcription activator. Apparently RNase A displays some properties of homologous angiogenin, which was shown to stimulate ribosomal RNA transcription and protein synthesis [46] and thus can possibly act as a transcriptional activator in tumour cells. B . The primary tumour site is a source of extracellular nucleoprotein complexes, in particular miRNA/Ago2 complexes [37] , [38] . Recent studies have shown a large number of small (∼20–30 nt) RNAs, originated from tRNAs, rRNA, snoRNAs and snRNAs, that are able to associate with Ago2 and perform the regulatory role by RNAi pathway [42] . In the bloodstream RNase A, whose concentration in the bloodstream is maintained by daily intramuscular injections, may generate short fragments of extracellular RNAs (tRNAs, rRNAs, snoRNAs, snRNAs), that may competitively displace miRNAs from nucleoprotein complexes, resulting in the degradation of released miRNAs by RNase A.
Figure Legend Snippet: Hypothetical mechanism of the effects of RNase A on the biogenesis of tumour-derived and circulating miRNAs. A . In tumour cells, positively charged RNase A binds with negatively charged tumour tissue (due to the presence of anion phosphatidylserine residues on the surface of tumour blood vessels) and penetrate into the cells. RNase A-mediated increase in miRNA levels in tumour cells may be a result of the activity of RNase A or its proteolytic fragment as a transcription activator. Apparently RNase A displays some properties of homologous angiogenin, which was shown to stimulate ribosomal RNA transcription and protein synthesis [46] and thus can possibly act as a transcriptional activator in tumour cells. B . The primary tumour site is a source of extracellular nucleoprotein complexes, in particular miRNA/Ago2 complexes [37] , [38] . Recent studies have shown a large number of small (∼20–30 nt) RNAs, originated from tRNAs, rRNA, snoRNAs and snRNAs, that are able to associate with Ago2 and perform the regulatory role by RNAi pathway [42] . In the bloodstream RNase A, whose concentration in the bloodstream is maintained by daily intramuscular injections, may generate short fragments of extracellular RNAs (tRNAs, rRNAs, snoRNAs, snRNAs), that may competitively displace miRNAs from nucleoprotein complexes, resulting in the degradation of released miRNAs by RNase A.

Techniques Used: Derivative Assay, Activity Assay, Activated Clotting Time Assay, Concentration Assay

Stem-loop RT-qPCR analysis of miRNA expression levels in tumour cells and blood serum of tumour-bearing mice after treatment with RNase A. A. miRNA expression in tumour tissue from mice with LLC that received RNase A therapy. The expression of miRNAs was normalised to U6 . B. miRNA levels in the blood serum of mice with LLC that received RNase A therapy. The concentration of serum miRNAs was normalised to serum volume. C. miRNA expression in LLC cells incubated in vitro with intact and DEPC-inactivated RNase A for 48 h. The expression of miRNAs was normalised to U6 and rpl30 . *, **, and *** denote a statistically significant difference relative to the control with p
Figure Legend Snippet: Stem-loop RT-qPCR analysis of miRNA expression levels in tumour cells and blood serum of tumour-bearing mice after treatment with RNase A. A. miRNA expression in tumour tissue from mice with LLC that received RNase A therapy. The expression of miRNAs was normalised to U6 . B. miRNA levels in the blood serum of mice with LLC that received RNase A therapy. The concentration of serum miRNAs was normalised to serum volume. C. miRNA expression in LLC cells incubated in vitro with intact and DEPC-inactivated RNase A for 48 h. The expression of miRNAs was normalised to U6 and rpl30 . *, **, and *** denote a statistically significant difference relative to the control with p

Techniques Used: Quantitative RT-PCR, Expressing, Mouse Assay, Concentration Assay, Incubation, In Vitro

Logarithmic bar-graph depiction of the results of sequencing data. The typical pattern of changes in the levels of some oncogenic and tumour-suppressor miRNAs ( A ), and members of the let-7 miRNA superfamily ( B ) in LLC tumour tissue and serum of tumour-bearing mice after RNase A therapy. Q corresponds to reads per kb per million (RPKM) = number of reads of specific miRNA/(size of miRNA(kb) ×total number of reads in the library(mln)). “−” – mice with LLC treated with saline buffer; “+” – mice with LLC treated with RNase A. Black and white bars reflect miRNA expression in tumour tissue, grey and spotted bars reflect miRNA level in the bloodstream.
Figure Legend Snippet: Logarithmic bar-graph depiction of the results of sequencing data. The typical pattern of changes in the levels of some oncogenic and tumour-suppressor miRNAs ( A ), and members of the let-7 miRNA superfamily ( B ) in LLC tumour tissue and serum of tumour-bearing mice after RNase A therapy. Q corresponds to reads per kb per million (RPKM) = number of reads of specific miRNA/(size of miRNA(kb) ×total number of reads in the library(mln)). “−” – mice with LLC treated with saline buffer; “+” – mice with LLC treated with RNase A. Black and white bars reflect miRNA expression in tumour tissue, grey and spotted bars reflect miRNA level in the bloodstream.

Techniques Used: Sequencing, Mouse Assay, Expressing

Scheme of the experiment for cDNA library preparation. Mice with intramuscularly (i.m.) implanted LLC were treated with saline or RNase A at a dose of 0.7 µg/kg for 10 days starting on the 4 th day after tumour transplantation. At 1 h after the last injection, tumour tissue and blood serum samples were collected and pooled according to groups, and long and short RNA fractions were isolated. Long RNA fractions RNA LTc and RNA LTR were used for qPCR for the evaluation of the expression levels of miRNA processing genes. Short tumour-derived (RNA STc and RNA STR ) and serum-derived (RNA SSc and RNA SSR ) RNA fractions were used for the preparation of cDNA libraries and subsequent sequencing on the SOLiD™ ABA 3.5 platform and for stem-loop qPCR.
Figure Legend Snippet: Scheme of the experiment for cDNA library preparation. Mice with intramuscularly (i.m.) implanted LLC were treated with saline or RNase A at a dose of 0.7 µg/kg for 10 days starting on the 4 th day after tumour transplantation. At 1 h after the last injection, tumour tissue and blood serum samples were collected and pooled according to groups, and long and short RNA fractions were isolated. Long RNA fractions RNA LTc and RNA LTR were used for qPCR for the evaluation of the expression levels of miRNA processing genes. Short tumour-derived (RNA STc and RNA STR ) and serum-derived (RNA SSc and RNA SSR ) RNA fractions were used for the preparation of cDNA libraries and subsequent sequencing on the SOLiD™ ABA 3.5 platform and for stem-loop qPCR.

Techniques Used: cDNA Library Assay, Mouse Assay, Transplantation Assay, Injection, Isolation, Real-time Polymerase Chain Reaction, Expressing, Derivative Assay, Sequencing

Heat map representing the miRNA profiles in the blood serum and tumour tissue in control and RNase A-treated mice. Dendrograms were derived by pairwise average linkage clustering of miRNA genes or samples using Euclidean distances between row-scaled or column-scaled RPKM values (Z-scores), respectively.
Figure Legend Snippet: Heat map representing the miRNA profiles in the blood serum and tumour tissue in control and RNase A-treated mice. Dendrograms were derived by pairwise average linkage clustering of miRNA genes or samples using Euclidean distances between row-scaled or column-scaled RPKM values (Z-scores), respectively.

Techniques Used: Mouse Assay, Derivative Assay

qPCR analysis of the expression of miRNA processing genes. A . Expression of miRNA processing genes in LLC tumour tissue from mice that received RNase A therapy. B . Expression of miRNA processing genes in LLC cells incubated in vitro with intact and DEPC-inactivated RNase A for 48 h. *, **, and *** denote a statistically significant difference relative to the control with p
Figure Legend Snippet: qPCR analysis of the expression of miRNA processing genes. A . Expression of miRNA processing genes in LLC tumour tissue from mice that received RNase A therapy. B . Expression of miRNA processing genes in LLC cells incubated in vitro with intact and DEPC-inactivated RNase A for 48 h. *, **, and *** denote a statistically significant difference relative to the control with p

Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Mouse Assay, Incubation, In Vitro

6) Product Images from "Determinants of R-loop formation at convergent bidirectionally transcribed trinucleotide repeats"

Article Title: Determinants of R-loop formation at convergent bidirectionally transcribed trinucleotide repeats

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkq935

RNA–DNA hybrid formation at a (CGG)39 · (CCG)39 FRAXA template. When the template DNA is transcribed with T7 RNA polymerase, heterogeneous RNA is produced generating a smear (Transcription lane). Treatment with RNase H alone which is specific to RNA base paired to template DNA digests only RNA that is base paired to its template DNA. Treatment with RNase A, which is specific to single-stranded RNA, digests all free, single-stranded RNA leaving template DNA and RNA:DNA hybrid structures. Note that RNA–DNA hybrids migrate more slowly than supercoiled DNA (as indicated schematically, RNA is in light blue). Hybrid structures generate a smear due to their heterogeneous sizes. With a larger RNA component, the DNA is open to a greater degree (more relaxed), hence migration is closer to open circular DNA. Treatment of the hybrids with RNase H along with RNase A removes any hybrids formed as well as transcript generated in the transcription reaction leaving only input template DNA. When transcription followed by RNase H or RNase A treatment alone or in combination is performed on an empty Bluescript vector [pBlueKS(+)], there is negligible hybrid formation.
Figure Legend Snippet: RNA–DNA hybrid formation at a (CGG)39 · (CCG)39 FRAXA template. When the template DNA is transcribed with T7 RNA polymerase, heterogeneous RNA is produced generating a smear (Transcription lane). Treatment with RNase H alone which is specific to RNA base paired to template DNA digests only RNA that is base paired to its template DNA. Treatment with RNase A, which is specific to single-stranded RNA, digests all free, single-stranded RNA leaving template DNA and RNA:DNA hybrid structures. Note that RNA–DNA hybrids migrate more slowly than supercoiled DNA (as indicated schematically, RNA is in light blue). Hybrid structures generate a smear due to their heterogeneous sizes. With a larger RNA component, the DNA is open to a greater degree (more relaxed), hence migration is closer to open circular DNA. Treatment of the hybrids with RNase H along with RNase A removes any hybrids formed as well as transcript generated in the transcription reaction leaving only input template DNA. When transcription followed by RNase H or RNase A treatment alone or in combination is performed on an empty Bluescript vector [pBlueKS(+)], there is negligible hybrid formation.

Techniques Used: Produced, Migration, Generated, Plasmid Preparation

Quantification of relative RNA–DNA hybrid formation at increasing repeat lengths. ( A ) In vitro transcription reactions were performed on FRAXA plasmids bearing repeat tracts of (CGG)17 · (CCG)17, (CGG)39 · (CCG)39 and (CGG)53 · (CCG)53 following which samples were treated with RNase A or A+H. Hybrid formation was quantified by densitometry analysis using image quant by measuring the proportion of products that migrate between open circular and supercoiled position (indicated by ‘R’) divided by the total products below open circular including supercoiled. RNase A+H treated samples were used to determine the position of the supercoiled DNA for each repeat length. To the left of the graph is a representative gel used to quantify relative hybrid formation. The RNA indicated in the graph represents the RNA component bound in the RNA:DNA hybrid. Error bars are derived from three separate experiments ( N =3). Using the t -test to compare R-loop formation revealed a significant difference between the 17 and 53 rCGG R-loop ( P =0.0413) as well as the 17 and 53 rCCG R-loop ( P =0.0092). ( B ) The same analysis was performed as in (A) but for SCA1 plasmids bearing repeat tracts of (CAG)30 · (CTG)30, (CAG) 49 · (CTG)49 and (CAG)74 · (CTG)74. Using the t -test to compare R-loop formation between each of the (CAG) · (CTG) repeat lengths did not reveal any statistically significant differences.
Figure Legend Snippet: Quantification of relative RNA–DNA hybrid formation at increasing repeat lengths. ( A ) In vitro transcription reactions were performed on FRAXA plasmids bearing repeat tracts of (CGG)17 · (CCG)17, (CGG)39 · (CCG)39 and (CGG)53 · (CCG)53 following which samples were treated with RNase A or A+H. Hybrid formation was quantified by densitometry analysis using image quant by measuring the proportion of products that migrate between open circular and supercoiled position (indicated by ‘R’) divided by the total products below open circular including supercoiled. RNase A+H treated samples were used to determine the position of the supercoiled DNA for each repeat length. To the left of the graph is a representative gel used to quantify relative hybrid formation. The RNA indicated in the graph represents the RNA component bound in the RNA:DNA hybrid. Error bars are derived from three separate experiments ( N =3). Using the t -test to compare R-loop formation revealed a significant difference between the 17 and 53 rCGG R-loop ( P =0.0413) as well as the 17 and 53 rCCG R-loop ( P =0.0092). ( B ) The same analysis was performed as in (A) but for SCA1 plasmids bearing repeat tracts of (CAG)30 · (CTG)30, (CAG) 49 · (CTG)49 and (CAG)74 · (CTG)74. Using the t -test to compare R-loop formation between each of the (CAG) · (CTG) repeat lengths did not reveal any statistically significant differences.

Techniques Used: In Vitro, Derivative Assay, CTG Assay

Effect of trinucleotide repeat interruptions on RNA–DNA hybrid formation. ( A ) In vitro transcription followed by RNase A or A + H treatment was performed with either pure (39p) FRAXA plasmids (CGG)39 · (CCG)39 or interrupted plasmids (39i) [(CGG)9(AGG) (CGG)9(AGG)(CGG)9(AGG)(CGG)9] · [(CCG)9(CCT) (CCG)9(CCT)(CCG)9(CCT)(CCG)9] as indicated. Repeat tract configurations are schematically presented where hollow dots are the CGG repeat units and the filled dots are the AGG interruptions. R-loops are indicated as ‘R’. ( B ) In vitro transcription followed by RNase A or A+H treatment was performed with either pure (49p) SCA1 plasmids (CAG)49 · (CTG)49 or interrupted plasmids (44i) [(CAG)12(CAT)(CAG)(CAT)(CAG)12(CAT)(CAG)(CAT)(CAG)14] · [(CTG)14(ATG)(CTG)(ATG)(CTG)12(ATG)(CAG)(ATG)(CTG)12] as indicated schematically where hollow dots are the CAG repeat units and the filled dots are the CAT interruptions.
Figure Legend Snippet: Effect of trinucleotide repeat interruptions on RNA–DNA hybrid formation. ( A ) In vitro transcription followed by RNase A or A + H treatment was performed with either pure (39p) FRAXA plasmids (CGG)39 · (CCG)39 or interrupted plasmids (39i) [(CGG)9(AGG) (CGG)9(AGG)(CGG)9(AGG)(CGG)9] · [(CCG)9(CCT) (CCG)9(CCT)(CCG)9(CCT)(CCG)9] as indicated. Repeat tract configurations are schematically presented where hollow dots are the CGG repeat units and the filled dots are the AGG interruptions. R-loops are indicated as ‘R’. ( B ) In vitro transcription followed by RNase A or A+H treatment was performed with either pure (49p) SCA1 plasmids (CAG)49 · (CTG)49 or interrupted plasmids (44i) [(CAG)12(CAT)(CAG)(CAT)(CAG)12(CAT)(CAG)(CAT)(CAG)14] · [(CTG)14(ATG)(CTG)(ATG)(CTG)12(ATG)(CAG)(ATG)(CTG)12] as indicated schematically where hollow dots are the CAG repeat units and the filled dots are the CAT interruptions.

Techniques Used: In Vitro, CTG Assay

Identification of R-loop structures formed in expanded DM1 (CTG)130 · (CAG)130 plasmids using EM following in vitro transcription and treatment with RNase A and SSB protein. SSB proteins bind to the looped-out non-template DNA in an R-loop structure. Thus, each R-loop structure is visualized as a loop within the DNA template as indicated by black arrowheads. ( A ) R-loops formed by using SP6 RNA polymerase, producing an rCAG:dCTG hybrid. ( B ) R-loops formed by using T7 RNA polymerase, producing an rCUG:dCAG hybrid.
Figure Legend Snippet: Identification of R-loop structures formed in expanded DM1 (CTG)130 · (CAG)130 plasmids using EM following in vitro transcription and treatment with RNase A and SSB protein. SSB proteins bind to the looped-out non-template DNA in an R-loop structure. Thus, each R-loop structure is visualized as a loop within the DNA template as indicated by black arrowheads. ( A ) R-loops formed by using SP6 RNA polymerase, producing an rCAG:dCTG hybrid. ( B ) R-loops formed by using T7 RNA polymerase, producing an rCUG:dCAG hybrid.

Techniques Used: CTG Assay, In Vitro

RNA–DNA hybrid formation during in vitro transcription of trinucleotide repeat-containing plasmids. ( A ) In vitro transcription of SCA1 plasmid containing (CAG)74 · (CTG)74 and FRAXA plasmid containing (CGG)39 · (CCG)39 repeats in either direction using T3 or T7 RNA polymerase. The repeat sequence contained within the RNA produced and bound in the hybrid is indicated below the transcribed template. Samples following transcription were subsequently treated with either RNase A or A+H as indicated to observe hybrid formation. R-loops are indicated on the gel as ‘R’. ( B ) Exact same reactions and gel conditions as in (A) but transcription was performed in the presence of 3.5 µCi [α- 32 P]-rCTP. Gel was dried and exposed to X-ray film as outlined in ‘Materials and Methods’ section.
Figure Legend Snippet: RNA–DNA hybrid formation during in vitro transcription of trinucleotide repeat-containing plasmids. ( A ) In vitro transcription of SCA1 plasmid containing (CAG)74 · (CTG)74 and FRAXA plasmid containing (CGG)39 · (CCG)39 repeats in either direction using T3 or T7 RNA polymerase. The repeat sequence contained within the RNA produced and bound in the hybrid is indicated below the transcribed template. Samples following transcription were subsequently treated with either RNase A or A+H as indicated to observe hybrid formation. R-loops are indicated on the gel as ‘R’. ( B ) Exact same reactions and gel conditions as in (A) but transcription was performed in the presence of 3.5 µCi [α- 32 P]-rCTP. Gel was dried and exposed to X-ray film as outlined in ‘Materials and Methods’ section.

Techniques Used: In Vitro, Plasmid Preparation, CTG Assay, Sequencing, Produced

Effect of convergent simultaneous bidirectional or serial transcription on R-loop formation. ( A ) In vitro transcription of FRAXA template (CGG)39 · (CCG)39 with either T3 or T7 RNA polymerase alone (rCCG or rCGG, respectively), or simultaneously (rCCG with rCGG) or serially: rCCG transcription then phenol chloroform extraction followed by rCGG transcription (rCCG then rCGG), and vice versa (rCGG then rCCG). R-loops are indicated as ‘R’. Note that in the case of bidirectional or serial transcription, complementary RNA is produced forming dsRNA as indicated on the gel by ‘*’. These products are not present in transcription reactions occurring in one direction. ( B ) Same as in (A) except in vitro transcription reactions were performed on a DM1 (CAG)79 · (CTG)79 template. ( C ) EM analysis of convergent transcription reaction products from DM1 (CAG)79 · (CTG)79 templates. Samples were transcribed convergently using T3 and T7 RNA polymerase promoters simultaneously then the products were treated with RNAse A and prepared for EM as described in the ‘Materials and Methods’ section (rCUGand rCAG RNase A). Samples were also subjected to RNase H treatment along with RNase A for comparison (rCUG and rCAG RNase A, H). Transcription was also performed on the same template in a single direction for further comparison (rCUG RNase A). The products observed for each type of transcription reaction is shown as a percentage of the total number of molecules analyzed. At least 100 molecules were analyzed for each type of transcription reaction.
Figure Legend Snippet: Effect of convergent simultaneous bidirectional or serial transcription on R-loop formation. ( A ) In vitro transcription of FRAXA template (CGG)39 · (CCG)39 with either T3 or T7 RNA polymerase alone (rCCG or rCGG, respectively), or simultaneously (rCCG with rCGG) or serially: rCCG transcription then phenol chloroform extraction followed by rCGG transcription (rCCG then rCGG), and vice versa (rCGG then rCCG). R-loops are indicated as ‘R’. Note that in the case of bidirectional or serial transcription, complementary RNA is produced forming dsRNA as indicated on the gel by ‘*’. These products are not present in transcription reactions occurring in one direction. ( B ) Same as in (A) except in vitro transcription reactions were performed on a DM1 (CAG)79 · (CTG)79 template. ( C ) EM analysis of convergent transcription reaction products from DM1 (CAG)79 · (CTG)79 templates. Samples were transcribed convergently using T3 and T7 RNA polymerase promoters simultaneously then the products were treated with RNAse A and prepared for EM as described in the ‘Materials and Methods’ section (rCUGand rCAG RNase A). Samples were also subjected to RNase H treatment along with RNase A for comparison (rCUG and rCAG RNase A, H). Transcription was also performed on the same template in a single direction for further comparison (rCUG RNase A). The products observed for each type of transcription reaction is shown as a percentage of the total number of molecules analyzed. At least 100 molecules were analyzed for each type of transcription reaction.

Techniques Used: In Vitro, Produced, CTG Assay

Mechanism of RNA:DNA hybrid formation during in vitro transcription. ( A ) Schematic of the two possible mechanisms for R-loop formation. By the thread-back model, the nascent transcript (depicted in light blue) that has been ejected from the RNA polymerase re-anneals with the complementary, free DNA template strand (depicted in red) following the progression of the RNA polymerase (light blue, oval). When transcription occurs in the presence of RNAse A (dark blue) the nascent transcript is degraded when it is ejected from the RNA polymerase hence cannot form the hybrid. By the extended-hybrid model, the nascent transcript remains bound to the template DNA and resists becoming ejected from the RNA polymerase. When transcribed in the presence of RNase A, as the nascent transcript is protected by being bound to the template DNA it is not degraded hence hybrid formation is not ablated. ( B ) FRAXA plasmid (CGG)39 · (CCG)39 transcribed in either direction in the absence (−) or presence (+) of RNase A during the transcription reaction. All transcription reactions were subjected to further RNase A or A + H treatment to analyze hybrid formation. ( C ) Same experiment as in (B) performed with SCA1 plasmid containing a (CAG)74 · (CTG)74 repeat tract.
Figure Legend Snippet: Mechanism of RNA:DNA hybrid formation during in vitro transcription. ( A ) Schematic of the two possible mechanisms for R-loop formation. By the thread-back model, the nascent transcript (depicted in light blue) that has been ejected from the RNA polymerase re-anneals with the complementary, free DNA template strand (depicted in red) following the progression of the RNA polymerase (light blue, oval). When transcription occurs in the presence of RNAse A (dark blue) the nascent transcript is degraded when it is ejected from the RNA polymerase hence cannot form the hybrid. By the extended-hybrid model, the nascent transcript remains bound to the template DNA and resists becoming ejected from the RNA polymerase. When transcribed in the presence of RNase A, as the nascent transcript is protected by being bound to the template DNA it is not degraded hence hybrid formation is not ablated. ( B ) FRAXA plasmid (CGG)39 · (CCG)39 transcribed in either direction in the absence (−) or presence (+) of RNase A during the transcription reaction. All transcription reactions were subjected to further RNase A or A + H treatment to analyze hybrid formation. ( C ) Same experiment as in (B) performed with SCA1 plasmid containing a (CAG)74 · (CTG)74 repeat tract.

Techniques Used: In Vitro, Plasmid Preparation, CTG Assay

7) Product Images from "Characterization of Intact Neo-Glycoproteins by Hydrophilic Interaction Liquid Chromatography "

Article Title: Characterization of Intact Neo-Glycoproteins by Hydrophilic Interaction Liquid Chromatography

Journal: Molecules

doi: 10.3390/molecules19079070

Graphical representation of the glycoform distributions (as abundance percentage) as calculated by relative abundance in MS spectra and peak heights (A) and peak areas (B) in HILIC-UV chromatograms for samples ( A ) Man-RNase A and ( B ) Ara(1→6)Man-RNase A.
Figure Legend Snippet: Graphical representation of the glycoform distributions (as abundance percentage) as calculated by relative abundance in MS spectra and peak heights (A) and peak areas (B) in HILIC-UV chromatograms for samples ( A ) Man-RNase A and ( B ) Ara(1→6)Man-RNase A.

Techniques Used: Mass Spectrometry, Hydrophilic Interaction Liquid Chromatography, Acetylene Reduction Assay

Monitoring of the synthesis of  neo -glycoconjugates by coupling Ara(1→6)Man-IME with RNase A.
Figure Legend Snippet: Monitoring of the synthesis of neo -glycoconjugates by coupling Ara(1→6)Man-IME with RNase A.

Techniques Used: Acetylene Reduction Assay

Chromatograms obtained applying the selected conditions (see experimental section) to the analyses of ( A ) sodium tetraborate buffer (100 mM, brought to pH 6 with HCl and diluted 1:2 with acetonitrile), ( B ) Man-IME (1 mg/mL in acetonitrile/water 50:50, v/v); ( C ) non-purified and ( D ) purified Man-RNase A (0.4 mg/mL in acetonitrile/water 50:50, v/v).
Figure Legend Snippet: Chromatograms obtained applying the selected conditions (see experimental section) to the analyses of ( A ) sodium tetraborate buffer (100 mM, brought to pH 6 with HCl and diluted 1:2 with acetonitrile), ( B ) Man-IME (1 mg/mL in acetonitrile/water 50:50, v/v); ( C ) non-purified and ( D ) purified Man-RNase A (0.4 mg/mL in acetonitrile/water 50:50, v/v).

Techniques Used: Purification

Representative chromatographic profiles for RNase A (red traces), RNase B (green traces) and their equimolar mixture (black traces) on TSKgel Amide-80, eluted at a flow rate of 0.2 mL/min. a Mobile phases: acetonitrile (solvent A) and water (solvent B) both containing 10 mM HClO 4 . Conditions: from 75 to 65% A in 20 min followed by isocratic elution at 65% A for 10 min. Injection volume: 2 µL. Column temperature: (panel A ) 25 °C and (panel B ) 50 °C.
Figure Legend Snippet: Representative chromatographic profiles for RNase A (red traces), RNase B (green traces) and their equimolar mixture (black traces) on TSKgel Amide-80, eluted at a flow rate of 0.2 mL/min. a Mobile phases: acetonitrile (solvent A) and water (solvent B) both containing 10 mM HClO 4 . Conditions: from 75 to 65% A in 20 min followed by isocratic elution at 65% A for 10 min. Injection volume: 2 µL. Column temperature: (panel A ) 25 °C and (panel B ) 50 °C.

Techniques Used: Flow Cytometry, Injection

( A ) Representative chromatograms of Ara(1→6)Man-RNase A and RNase A (1 mg/mL and 0.25 mg/mL, respectively; acetonitrile/water 50:50, v/v) obtained applying the selected chromatographic conditions (see experimental section). ( B ) Deconvoluted ESI-LTQ-MS spectrum for RNase A (13,681 Da). ( C ) Deconvoluted ESI-LTQ-MS spectrum for Ara(1→6)Man-RNase A (RNase A 13,681 Da + 367 Da per Ara(1→6)Man unit added).
Figure Legend Snippet: ( A ) Representative chromatograms of Ara(1→6)Man-RNase A and RNase A (1 mg/mL and 0.25 mg/mL, respectively; acetonitrile/water 50:50, v/v) obtained applying the selected chromatographic conditions (see experimental section). ( B ) Deconvoluted ESI-LTQ-MS spectrum for RNase A (13,681 Da). ( C ) Deconvoluted ESI-LTQ-MS spectrum for Ara(1→6)Man-RNase A (RNase A 13,681 Da + 367 Da per Ara(1→6)Man unit added).

Techniques Used: Acetylene Reduction Assay, Mass Spectrometry

( A ) Representative chromatograms of Man-RNase A and RNase A (1 mg/mL and 0.25 mg/mL, respectively; acetonitrile/water 50:50, v/v) obtained applying the selected chromatographic conditions (see experimental section). ( B ) Deconvoluted ESI-LTQ-MS spectrum for RNase A (13,681 Da). ( C ) Deconvoluted ESI-LTQ-MS spectrum for Man-RNase A (RNase A 13,681 Da, + 235 Da per mannose unit added).
Figure Legend Snippet: ( A ) Representative chromatograms of Man-RNase A and RNase A (1 mg/mL and 0.25 mg/mL, respectively; acetonitrile/water 50:50, v/v) obtained applying the selected chromatographic conditions (see experimental section). ( B ) Deconvoluted ESI-LTQ-MS spectrum for RNase A (13,681 Da). ( C ) Deconvoluted ESI-LTQ-MS spectrum for Man-RNase A (RNase A 13,681 Da, + 235 Da per mannose unit added).

Techniques Used: Mass Spectrometry

Synthesis of  neo -glycoproteins by coupling of IME-thioglycoside with RNase A.
Figure Legend Snippet: Synthesis of neo -glycoproteins by coupling of IME-thioglycoside with RNase A.

Techniques Used:

8) Product Images from "Maternal RNA regulates Aurora C kinase during mouse oocyte maturation in a translation-independent fashion †"

Article Title: Maternal RNA regulates Aurora C kinase during mouse oocyte maturation in a translation-independent fashion †

Journal: Biology of Reproduction

doi: 10.1093/biolre/iox047

Maternal RNA perturbs bipolar spindle assembly. Full-grown, prophase I-arrested oocytes were injected with PBS or RNase A followed by in vitro maturation to Met I. (A) Met I oocytes were fixed and stained with an anti-α-tubulin antibody to label spindle microtubules (green) and DAPI to label DNA (red) followed by confocal imaging. Representative images are shown. Scale bar represents 10 μm. (B) Quantification of abnormal spindle morphology (C) Quantification of spindle length/width ratio. (D) Quantification of the number of oocytes with a monopolar spindle. (E) Quantifications of oocytes that failed to form a spindle. (F) Quantification of the number of oocytes with misaligned chromosomes. The experiment was carried out three times, and the total number of oocytes examined were 30 and 34 oocytes in the control and RNase A groups, respectively. The data are expressed as mean ± SEM and Student t -test was used to analyze the data. Values with asterisks vary significantly, * P
Figure Legend Snippet: Maternal RNA perturbs bipolar spindle assembly. Full-grown, prophase I-arrested oocytes were injected with PBS or RNase A followed by in vitro maturation to Met I. (A) Met I oocytes were fixed and stained with an anti-α-tubulin antibody to label spindle microtubules (green) and DAPI to label DNA (red) followed by confocal imaging. Representative images are shown. Scale bar represents 10 μm. (B) Quantification of abnormal spindle morphology (C) Quantification of spindle length/width ratio. (D) Quantification of the number of oocytes with a monopolar spindle. (E) Quantifications of oocytes that failed to form a spindle. (F) Quantification of the number of oocytes with misaligned chromosomes. The experiment was carried out three times, and the total number of oocytes examined were 30 and 34 oocytes in the control and RNase A groups, respectively. The data are expressed as mean ± SEM and Student t -test was used to analyze the data. Values with asterisks vary significantly, * P

Techniques Used: Injection, In Vitro, Staining, Imaging

Maternal RNA perturbs AURKC localization during oocyte maturation. Full-grown prophase I-arrested oocytes were injected with PBS or RNase A followed by in vitro maturation. (A) Met I oocytes (6 h) were fixed and immunostained with an anti-AURKC antibody (green in merge). DNA was detected with DAPI (blue in merge). (B) Corresponding quantification of oocytes with localized AURKC in (A). (C) Telophase I oocytes were fixed and immunostained with an anti-AURKC antibody (red in merge) and an anti-α-tubulin antibody (green in merge). DNA was labeled with DAPI (blue in merge). The scale bar represents 10 μm, and representative images are shown. The experiments were carried out two times and the total number of oocytes examined were 20 and 24 oocytes in the control and RNase A groups, respectively. The data are expressed as mean ± SEM, and Student t -test was used to analyze the data. Values with asterisks vary significantly, *** P
Figure Legend Snippet: Maternal RNA perturbs AURKC localization during oocyte maturation. Full-grown prophase I-arrested oocytes were injected with PBS or RNase A followed by in vitro maturation. (A) Met I oocytes (6 h) were fixed and immunostained with an anti-AURKC antibody (green in merge). DNA was detected with DAPI (blue in merge). (B) Corresponding quantification of oocytes with localized AURKC in (A). (C) Telophase I oocytes were fixed and immunostained with an anti-AURKC antibody (red in merge) and an anti-α-tubulin antibody (green in merge). DNA was labeled with DAPI (blue in merge). The scale bar represents 10 μm, and representative images are shown. The experiments were carried out two times and the total number of oocytes examined were 20 and 24 oocytes in the control and RNase A groups, respectively. The data are expressed as mean ± SEM, and Student t -test was used to analyze the data. Values with asterisks vary significantly, *** P

Techniques Used: Injection, In Vitro, Labeling

Maternal RNA perturbs CPC localization and activity during oocyte maturation. Full-grown prophase I-arrested oocytes were injected with PBS or RNase A followed by in vitro maturation to Met I. Met I oocytes (6 h) were fixed and immunostained (green in merge) with an anti-survivin antibody (A), anti-pINCENP antibody (C), and anti-H3pS10 antibody (E). DNA was detected with DAPI (blue in merge). Representative images are shown, scale bar represents 10 μm. (B), (D) and (F) Corresponding quantifications of fluorescence intensities of (A), (C) and (E), respectively. The experiments were carried out two times and the total numbers of examined oocytes are indicated above the graph bars. The data are expressed as mean ± SEM, and Student t -test was used to analyze the data. Values with asterisks vary significantly, * P
Figure Legend Snippet: Maternal RNA perturbs CPC localization and activity during oocyte maturation. Full-grown prophase I-arrested oocytes were injected with PBS or RNase A followed by in vitro maturation to Met I. Met I oocytes (6 h) were fixed and immunostained (green in merge) with an anti-survivin antibody (A), anti-pINCENP antibody (C), and anti-H3pS10 antibody (E). DNA was detected with DAPI (blue in merge). Representative images are shown, scale bar represents 10 μm. (B), (D) and (F) Corresponding quantifications of fluorescence intensities of (A), (C) and (E), respectively. The experiments were carried out two times and the total numbers of examined oocytes are indicated above the graph bars. The data are expressed as mean ± SEM, and Student t -test was used to analyze the data. Values with asterisks vary significantly, * P

Techniques Used: Activity Assay, Injection, In Vitro, Fluorescence

Depletion of maternal RNA impairs oocyte maturation. Full-grown prophase I-arrested oocytes were injected with PBS or RNase A followed by maturation in vitro for 16 h. (A) First polar body extrusion (PBE) was scored to assess meiotic progression. (B) Quantification of cytokinesis defects based on live cell imaging (n = 21, 26). (C) Representative live cell images showing retraction of the extruded polar body in RNA-depleted oocytes; scale bar represents 100 μm. White arrowheads in the lower panel indicates chromatin aggregation; scale bar, 10 μm. (D) Confocal microscopy images of Met II eggs stained with an anti-α-tubulin antibody to label spindle (green) and DAPI to label DNA (red). Representative images are shown. (E) Quantification of chromosomal collapse phenotype. The experiments were carried out at least three times and the total numbers of oocytes examined are indicated above the graph bars. The data are expressed as mean ± SEM, and Student t -test was used to analyze the data except (A) where one-way ANOVA was used to analyze the data. Values with asterisks vary significantly, * P
Figure Legend Snippet: Depletion of maternal RNA impairs oocyte maturation. Full-grown prophase I-arrested oocytes were injected with PBS or RNase A followed by maturation in vitro for 16 h. (A) First polar body extrusion (PBE) was scored to assess meiotic progression. (B) Quantification of cytokinesis defects based on live cell imaging (n = 21, 26). (C) Representative live cell images showing retraction of the extruded polar body in RNA-depleted oocytes; scale bar represents 100 μm. White arrowheads in the lower panel indicates chromatin aggregation; scale bar, 10 μm. (D) Confocal microscopy images of Met II eggs stained with an anti-α-tubulin antibody to label spindle (green) and DAPI to label DNA (red). Representative images are shown. (E) Quantification of chromosomal collapse phenotype. The experiments were carried out at least three times and the total numbers of oocytes examined are indicated above the graph bars. The data are expressed as mean ± SEM, and Student t -test was used to analyze the data except (A) where one-way ANOVA was used to analyze the data. Values with asterisks vary significantly, * P

Techniques Used: Injection, In Vitro, Live Cell Imaging, Confocal Microscopy, Staining

Regulation of AURKC and meiotic spindle by maternal RNA during oocyte maturation is independent of translation. Full-grown prophase I-arrested oocytes were matured in vitro for 6 h (Met I) or 14 h (Met II). Met I and Met II oocytes were injected with PBS or RNase A followed by incubation in the same maturation condition for an additional 2 h. Met I (A-C) and Met II (D-F) injected oocytes were fixed and immunostained with an anti-AURKC antibody (red in merge) and an anti-α-tubulin antibody (green in merge). DNA was labeled with DAPI (blue in merge). The scale bar represents 10 μm, and representative images are shown. (B, C) Corresponding quantifications of oocytes in A. (E, F) Corresponding quantifications of eggs from D. The experiments were carried out two times and the total numbers of examined oocytes are indicated above the graph bars. The data are expressed as mean ± SEM, and Student t -test was used to analyze the data. Values with asterisks vary significantly, * P
Figure Legend Snippet: Regulation of AURKC and meiotic spindle by maternal RNA during oocyte maturation is independent of translation. Full-grown prophase I-arrested oocytes were matured in vitro for 6 h (Met I) or 14 h (Met II). Met I and Met II oocytes were injected with PBS or RNase A followed by incubation in the same maturation condition for an additional 2 h. Met I (A-C) and Met II (D-F) injected oocytes were fixed and immunostained with an anti-AURKC antibody (red in merge) and an anti-α-tubulin antibody (green in merge). DNA was labeled with DAPI (blue in merge). The scale bar represents 10 μm, and representative images are shown. (B, C) Corresponding quantifications of oocytes in A. (E, F) Corresponding quantifications of eggs from D. The experiments were carried out two times and the total numbers of examined oocytes are indicated above the graph bars. The data are expressed as mean ± SEM, and Student t -test was used to analyze the data. Values with asterisks vary significantly, * P

Techniques Used: In Vitro, Injection, Incubation, Labeling

9) Product Images from "Analysis of the oligomeric states of nucleophosmin using size exclusion chromatography"

Article Title: Analysis of the oligomeric states of nucleophosmin using size exclusion chromatography

Journal: Scientific Reports

doi: 10.1038/s41598-018-22359-w

Association of NPM1 with RNA in the high molecular weight fractions. ( A ) Triton X-100 soluble and insoluble cell lysates were subjected to SEC. The indicated fractions were pooled, then immunoprecipitated with anti-NPM. NPM1 and RNA were detected by immunoblot and ethidium bromide staining, respectively. ( B ) HeLa cell lysates prepared as in Fig.   2A  were treated with or without RNase A, then subjected to SEC. Fractions containing the four NPM1 groups identified in Fig.   3A  are underlined.
Figure Legend Snippet: Association of NPM1 with RNA in the high molecular weight fractions. ( A ) Triton X-100 soluble and insoluble cell lysates were subjected to SEC. The indicated fractions were pooled, then immunoprecipitated with anti-NPM. NPM1 and RNA were detected by immunoblot and ethidium bromide staining, respectively. ( B ) HeLa cell lysates prepared as in Fig.  2A were treated with or without RNase A, then subjected to SEC. Fractions containing the four NPM1 groups identified in Fig.  3A are underlined.

Techniques Used: Molecular Weight, Size-exclusion Chromatography, Immunoprecipitation, Staining

The main component of cellular NPM1 complex is NPM1 and its variant. Both the Triton X-100 soluble and insoluble lysates from HeLa cells were subjected to SEC. Fractions 15 and 16 from the soluble sample (for Group 1), fractions 19 to 21 from the soluble sample (for Group 2), and fractions 15 and 16 from the insoluble sample (for Group 3) were separately pooled. After treatment with or without RNase A, each pooled sample was immunoprecipitated with anti-NPM antibody. Proteins were detected by CBB staining ( Upper panel ) and immunoblot using anti-NPM antibody ( Lower panel ).
Figure Legend Snippet: The main component of cellular NPM1 complex is NPM1 and its variant. Both the Triton X-100 soluble and insoluble lysates from HeLa cells were subjected to SEC. Fractions 15 and 16 from the soluble sample (for Group 1), fractions 19 to 21 from the soluble sample (for Group 2), and fractions 15 and 16 from the insoluble sample (for Group 3) were separately pooled. After treatment with or without RNase A, each pooled sample was immunoprecipitated with anti-NPM antibody. Proteins were detected by CBB staining ( Upper panel ) and immunoblot using anti-NPM antibody ( Lower panel ).

Techniques Used: Variant Assay, Size-exclusion Chromatography, Immunoprecipitation, Staining

Elution profiles of endogenous NPM1 in HeLa cells. ( A ) ( Upper panel ) Elution profiles of Triton X-100 soluble NPM1 and RNA. ( Lower panel ) Elution profiles of Triton X-100 insoluble NPM1 and RNA. Endogenous NPM1 was detected by immunoblot. Protein mass standards are indicated above the  panel : thyroglobulin (669 kDa), ferritin (440 kDa), aldolase (158 kDa), conalbumin (75 kDa), ovalbumin (44 kDa), and Ribonuclease A (13.7 kDa). RNA was detected by ethidium bromide staining. The four NPM1 groups (Group 1 to Group 4) identified by SEC are underlined. ( B ) Schematic representation of sample preparation in C. ( C ) Elution profile of sonication-treated Triton X-100 soluble NPM1 prepared in B.
Figure Legend Snippet: Elution profiles of endogenous NPM1 in HeLa cells. ( A ) ( Upper panel ) Elution profiles of Triton X-100 soluble NPM1 and RNA. ( Lower panel ) Elution profiles of Triton X-100 insoluble NPM1 and RNA. Endogenous NPM1 was detected by immunoblot. Protein mass standards are indicated above the panel : thyroglobulin (669 kDa), ferritin (440 kDa), aldolase (158 kDa), conalbumin (75 kDa), ovalbumin (44 kDa), and Ribonuclease A (13.7 kDa). RNA was detected by ethidium bromide staining. The four NPM1 groups (Group 1 to Group 4) identified by SEC are underlined. ( B ) Schematic representation of sample preparation in C. ( C ) Elution profile of sonication-treated Triton X-100 soluble NPM1 prepared in B.

Techniques Used: Staining, Size-exclusion Chromatography, Sample Prep, Sonication

Elution profiles of NPM1 monomer and oligomers expressed in cells. 293T cells were transfected with expression vector for G196-NPM1 (WT) or G196-NPM1 (LG). ( A ) The Triton X-100 soluble lysates were used for immunoprecipitation with anti-G196 antibody. G196-NPM1 and extracted RNA were detected by immunoblot and ethidium bromide staining, respectively. ( B ) Interaction of G196-NPM (WT) or G196-NPM1 (LG) with endogenous NPM1. The Triton X-100 soluble (S) and insoluble (I) cell lysates were treated with RNase A, then immunoprecipitated with anti-G196 antibody. ( C ) Elution profiles of G196-NPM1 (WT) in the Triton X-100 soluble and insoluble cell lysates. Fractions containing the four NPM1 groups identified in Fig.   3A  are underlined. ( D ) Elution profile of G196-NPM1 (LG) in the Triton X-100-soluble cell lysates. ( E ) Elution profiles of G196-NPM1 (WT) in the RNase A-treated Triton X-100 soluble and insoluble cell lysates. Fractions containing the four NPM1 groups identified in Fig.   3A  were underlined.
Figure Legend Snippet: Elution profiles of NPM1 monomer and oligomers expressed in cells. 293T cells were transfected with expression vector for G196-NPM1 (WT) or G196-NPM1 (LG). ( A ) The Triton X-100 soluble lysates were used for immunoprecipitation with anti-G196 antibody. G196-NPM1 and extracted RNA were detected by immunoblot and ethidium bromide staining, respectively. ( B ) Interaction of G196-NPM (WT) or G196-NPM1 (LG) with endogenous NPM1. The Triton X-100 soluble (S) and insoluble (I) cell lysates were treated with RNase A, then immunoprecipitated with anti-G196 antibody. ( C ) Elution profiles of G196-NPM1 (WT) in the Triton X-100 soluble and insoluble cell lysates. Fractions containing the four NPM1 groups identified in Fig.  3A are underlined. ( D ) Elution profile of G196-NPM1 (LG) in the Triton X-100-soluble cell lysates. ( E ) Elution profiles of G196-NPM1 (WT) in the RNase A-treated Triton X-100 soluble and insoluble cell lysates. Fractions containing the four NPM1 groups identified in Fig.  3A were underlined.

Techniques Used: Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Staining

Purified recombinant NPM1 shows a similar elution profile to that of RNase A-treated cellular NPM1. ( A)  Bacterially expressed His-TEV-NPM1 was purified as described in experimental procedures. Purified proteins showed high purity and did not contain any RNA. ( B)  Elution profile of purified His-TEV-NPM1 proteins.
Figure Legend Snippet: Purified recombinant NPM1 shows a similar elution profile to that of RNase A-treated cellular NPM1. ( A) Bacterially expressed His-TEV-NPM1 was purified as described in experimental procedures. Purified proteins showed high purity and did not contain any RNA. ( B) Elution profile of purified His-TEV-NPM1 proteins.

Techniques Used: Purification, Recombinant

10) Product Images from "Nickel affects xylem Sap RNase a and converts RNase A to a urease"

Article Title: Nickel affects xylem Sap RNase a and converts RNase A to a urease

Journal: BMC Plant Biology

doi: 10.1186/1471-2229-13-207

The N-terminal sequence and alignment of 14-kDa pecan sap RNase A with reported proteins. (A) 14-kDa pecan sap RNase A. The matching of the sequence alignment (within 29 amino acid residues) is 96%. (B) Bovine pancreatic RNase A. The matching of the sequence alignment (N-terminal first 29 amino acid residues) is 96%.
Figure Legend Snippet: The N-terminal sequence and alignment of 14-kDa pecan sap RNase A with reported proteins. (A) 14-kDa pecan sap RNase A. The matching of the sequence alignment (within 29 amino acid residues) is 96%. (B) Bovine pancreatic RNase A. The matching of the sequence alignment (N-terminal first 29 amino acid residues) is 96%.

Techniques Used: Sequencing

Bovine pancreatic RNase A catalyzing the hydrolysis of urea. Net absorbance at 340 nm was scanned after adding bovine pancreatic RNase A [6.7 μg protein (upper line) and 20 μg protein (lower line)] into the reaction mixture for urease activity assay. Data indicates a positive correlation between the amounts of RNase A added to the reaction mixture and the breakdown of urea. A very similar graph was obtained with pecan sap RNase A catalyzing the hydrolysis of urea.
Figure Legend Snippet: Bovine pancreatic RNase A catalyzing the hydrolysis of urea. Net absorbance at 340 nm was scanned after adding bovine pancreatic RNase A [6.7 μg protein (upper line) and 20 μg protein (lower line)] into the reaction mixture for urease activity assay. Data indicates a positive correlation between the amounts of RNase A added to the reaction mixture and the breakdown of urea. A very similar graph was obtained with pecan sap RNase A catalyzing the hydrolysis of urea.

Techniques Used: Activity Assay

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of bovine pancreatic and pecan xylem RNase A. (A)  Bovine pancreatic RNase A. Approximately 200 μg of protein was loaded. STD: Standard (loaded: 12 μl). Numbers in the left and right columns of the figure indicate molecular mass.  (B)  Purified pecan urease. The 14-kDa enzyme has been purified to homogeneity. Numbers in the left column and right side of the figure indicate molecular mass in kDa. The migration distance on the SDS-PAGE gel indicates the size of protein. Std = Protein Standards.
Figure Legend Snippet: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of bovine pancreatic and pecan xylem RNase A. (A) Bovine pancreatic RNase A. Approximately 200 μg of protein was loaded. STD: Standard (loaded: 12 μl). Numbers in the left and right columns of the figure indicate molecular mass. (B) Purified pecan urease. The 14-kDa enzyme has been purified to homogeneity. Numbers in the left column and right side of the figure indicate molecular mass in kDa. The migration distance on the SDS-PAGE gel indicates the size of protein. Std = Protein Standards.

Techniques Used: Polyacrylamide Gel Electrophoresis, Purification, Migration, SDS Page

Cleavage of RNA with bovine pancreatic 13.4 kDa protein. Cleaving RNA with the pecan sap 14 kDa RNase A protein produces a similar graph.
Figure Legend Snippet: Cleavage of RNA with bovine pancreatic 13.4 kDa protein. Cleaving RNA with the pecan sap 14 kDa RNase A protein produces a similar graph.

Techniques Used:

Alignment BLAST/NCBI: Search results for short, nearly exact matches of bovine pancreatic RNase A (13,473 Da; total 124 amino acid residues) and urease (total 567 amino acid residues) from Psychrobacter cryohalolentis K5. A . Identities = 4/5 (80%); B . Identities = 4/5 (80%); C . Identities = 4/4 (100%); D . Identities = 5/5 (100%); E . Identities = 4/9 (44%); F . Identities = 8/11 (72%); G . Identities = 3/4 (75%).
Figure Legend Snippet: Alignment BLAST/NCBI: Search results for short, nearly exact matches of bovine pancreatic RNase A (13,473 Da; total 124 amino acid residues) and urease (total 567 amino acid residues) from Psychrobacter cryohalolentis K5. A . Identities = 4/5 (80%); B . Identities = 4/5 (80%); C . Identities = 4/4 (100%); D . Identities = 5/5 (100%); E . Identities = 4/9 (44%); F . Identities = 8/11 (72%); G . Identities = 3/4 (75%).

Techniques Used:

11) Product Images from "A phosphate-binding subsite in bovine pancreatic ribonuclease A can be converted into a very efficient catalytic site"

Article Title: A phosphate-binding subsite in bovine pancreatic ribonuclease A can be converted into a very efficient catalytic site

Journal: Protein Science : A Publication of the Protein Society

doi: 10.1110/ps.062251707

( A ) Three-dimensional structure of RNase A. Amino acid residues belonging to the active site p 1  (His12, His 119, and Lys41) and to the noncatalytic phosphate-binding subsites p 0  (Lys66) and p 2  (Lys7 and Arg10) are shown in stick. The picture was obtained
Figure Legend Snippet: ( A ) Three-dimensional structure of RNase A. Amino acid residues belonging to the active site p 1 (His12, His 119, and Lys41) and to the noncatalytic phosphate-binding subsites p 0 (Lys66) and p 2 (Lys7 and Arg10) are shown in stick. The picture was obtained

Techniques Used: Binding Assay

Positive and negative controls of recombinant RNases. Activities were determined by the zymogram technique in 15% SDS-PAGE containing poly(C) as substrate. (Lane  1 ) 300 pg of RNase A. (Lane  2 ) 5 μl of the intracellular soluble fraction of  E. coli
Figure Legend Snippet: Positive and negative controls of recombinant RNases. Activities were determined by the zymogram technique in 15% SDS-PAGE containing poly(C) as substrate. (Lane 1 ) 300 pg of RNase A. (Lane 2 ) 5 μl of the intracellular soluble fraction of E. coli

Techniques Used: Recombinant, SDS Page

SDS-15% PAGE with Coomassie blue staining ( A ) and RNase activity staining on gels containing either poly(C) ( B ) or poly(U) ( C ) as substrates. ( A ) (Lane  1 ) native RNase A. (Lane  2 ) H12K/H119Q-RNase A. (Lane  3 ) K7H/R10H/H12K/H119Q-RNase A. (Lane  4 ) K7H/R10H-RNase
Figure Legend Snippet: SDS-15% PAGE with Coomassie blue staining ( A ) and RNase activity staining on gels containing either poly(C) ( B ) or poly(U) ( C ) as substrates. ( A ) (Lane 1 ) native RNase A. (Lane 2 ) H12K/H119Q-RNase A. (Lane 3 ) K7H/R10H/H12K/H119Q-RNase A. (Lane 4 ) K7H/R10H-RNase

Techniques Used: Polyacrylamide Gel Electrophoresis, Staining, Activity Assay

Three-dimensional structure of the region around the RNase A active site. ( A ) RNase A. ( B , C ) Molecular modeling of the variants K7H/R10H-RNase A and K7H/R10H/H12K/H119Q-RNase A, respectively. Molecular modeling was carried out with the program Deep View/Swiss
Figure Legend Snippet: Three-dimensional structure of the region around the RNase A active site. ( A ) RNase A. ( B , C ) Molecular modeling of the variants K7H/R10H-RNase A and K7H/R10H/H12K/H119Q-RNase A, respectively. Molecular modeling was carried out with the program Deep View/Swiss

Techniques Used:

Effect of the presence of a new active site on the exonucleolytic versus endonucleolytic activity of RNase A. Tetranucleotide/dinucleotide ratio for the cleavage of the pentacytidylic acid substrate (Cp) 4 C > p by K7H/R10H-RNase A ( A ) and by the
Figure Legend Snippet: Effect of the presence of a new active site on the exonucleolytic versus endonucleolytic activity of RNase A. Tetranucleotide/dinucleotide ratio for the cleavage of the pentacytidylic acid substrate (Cp) 4 C > p by K7H/R10H-RNase A ( A ) and by the

Techniques Used: Activity Assay

Analysis by reversed-phase HPLC of the poly(C) substrate before addition of enzyme ( A ) and of the products obtained by digestion with RNase A ( B ) (the oligonucleotide size of the products is indicated). ( C ) Comparison of oligocytidilyc acid formation
Figure Legend Snippet: Analysis by reversed-phase HPLC of the poly(C) substrate before addition of enzyme ( A ) and of the products obtained by digestion with RNase A ( B ) (the oligonucleotide size of the products is indicated). ( C ) Comparison of oligocytidilyc acid formation

Techniques Used: High Performance Liquid Chromatography

12) Product Images from "Picornavirus RNA is protected from cleavage by ribonuclease during virion uncoating and transfer across cellular and model membranes"

Article Title: Picornavirus RNA is protected from cleavage by ribonuclease during virion uncoating and transfer across cellular and model membranes

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1006197

PV infectivity is not affected by covalent linkage of RNase A to the virus. A) RNase A is covalently linked to PV VP1. Conjugation reactions containing 35 S-Met/Cys radiolabelled PV and/or RNase A (as indicated at the top of the image) were subjected to SDS-PAGE and western blot with antisera against RNase A. The major over-exposed bands correspond to RNase A monomer and dimer (indicated by arrows). Bands in the middle lane are the expected size for radiolabelled virus proteins VP1, VP2 and VP3. The upper band in the left hand lane is the expected size for RNase A covalently attached to VP1 (as indicated by arrow). Molecular weight standards (kDa) are shown on the left. B) Plaque assay of PV conjugated to RNase A (0, 90, 300, 600 molar ratio). Plaque forming units (pfu) were expressed as percentage of no RNase A control and data pooled from three independent experiments. C) Ribonuclease activity was measured by quantifying tRNA fluorescence (Relative Fluorescence Units, RFU) with Ribogreen in the presence of RNase A, purified PV, PV conjugated to RNase A with EDC (PV-A + EDC) and mock conjugation reaction (PV-A—EDC). D) Sucrose gradient profile of 3 H-U-PV RNA (as in Fig 3E ) uncoated in vitro at 50°C for 10 min in the presence of RNase A (1mg/ml) or PBS carrier control (representative of two independent experiments). E) Sucrose gradient profile (as in Fig 3E ) of 3 H-U-PV RNA from viral particles directly conjugated to RNase A with the cross-linker EDC (PV-A + EDC) or mock conjugated (no EDC, PV-A—EDC) uncoated in vitro at 50°C for 10 min (representative of two independent experiments). F) Representative image of HeLa Ohio cells infected with PV conjugated to Cy2 (green, left panel) and RNase A-DyLight594 (red, middle panel) fixed 15 min post infection. The degree of co-internalization (Merge, right panel) was measured for 10 random cells (R = 0.92 +/- 0.06 (SD). Nuclei were stained with Hoechst (blue). Scale bar is 5 μm. G) RNase activity (as in C) of individual and mixed components of the RNase S system. H) RNase activity (as in C and G) and I) virus titre (as in B) of PV conjugated to individual or mixed components of the RNase S system. (All data from three independent experiments with error bars showing standard error, unless stated).
Figure Legend Snippet: PV infectivity is not affected by covalent linkage of RNase A to the virus. A) RNase A is covalently linked to PV VP1. Conjugation reactions containing 35 S-Met/Cys radiolabelled PV and/or RNase A (as indicated at the top of the image) were subjected to SDS-PAGE and western blot with antisera against RNase A. The major over-exposed bands correspond to RNase A monomer and dimer (indicated by arrows). Bands in the middle lane are the expected size for radiolabelled virus proteins VP1, VP2 and VP3. The upper band in the left hand lane is the expected size for RNase A covalently attached to VP1 (as indicated by arrow). Molecular weight standards (kDa) are shown on the left. B) Plaque assay of PV conjugated to RNase A (0, 90, 300, 600 molar ratio). Plaque forming units (pfu) were expressed as percentage of no RNase A control and data pooled from three independent experiments. C) Ribonuclease activity was measured by quantifying tRNA fluorescence (Relative Fluorescence Units, RFU) with Ribogreen in the presence of RNase A, purified PV, PV conjugated to RNase A with EDC (PV-A + EDC) and mock conjugation reaction (PV-A—EDC). D) Sucrose gradient profile of 3 H-U-PV RNA (as in Fig 3E ) uncoated in vitro at 50°C for 10 min in the presence of RNase A (1mg/ml) or PBS carrier control (representative of two independent experiments). E) Sucrose gradient profile (as in Fig 3E ) of 3 H-U-PV RNA from viral particles directly conjugated to RNase A with the cross-linker EDC (PV-A + EDC) or mock conjugated (no EDC, PV-A—EDC) uncoated in vitro at 50°C for 10 min (representative of two independent experiments). F) Representative image of HeLa Ohio cells infected with PV conjugated to Cy2 (green, left panel) and RNase A-DyLight594 (red, middle panel) fixed 15 min post infection. The degree of co-internalization (Merge, right panel) was measured for 10 random cells (R = 0.92 +/- 0.06 (SD). Nuclei were stained with Hoechst (blue). Scale bar is 5 μm. G) RNase activity (as in C) of individual and mixed components of the RNase S system. H) RNase activity (as in C and G) and I) virus titre (as in B) of PV conjugated to individual or mixed components of the RNase S system. (All data from three independent experiments with error bars showing standard error, unless stated).

Techniques Used: Infection, Conjugation Assay, SDS Page, Western Blot, Molecular Weight, Plaque Assay, Activity Assay, Fluorescence, Purification, In Vitro, Staining

ERAV is co-internalized with RNase A but infectivity is not compromised. A) Representative images of HeLa Ohio cells infected with ERAV conjugated to Cy2 (green, left panel) and RNase A-DyLight594 (red, middle panel) fixed 15 min post infection. The degree of co-internalization (Merge, right panel) was measured for 10 random cells (R = 0.86 +/- 0.09 (SD). Nuclei were stained with Hoechst (blue). Scale bar is 5 μm. B) Plaque assay of ERAV in the presence of 0–1 mg/ml RNase A. Plaque forming units (pfu) were expressed as percentage of no RNase A control. Data are from three independent experiments with error bars showing standard error.
Figure Legend Snippet: ERAV is co-internalized with RNase A but infectivity is not compromised. A) Representative images of HeLa Ohio cells infected with ERAV conjugated to Cy2 (green, left panel) and RNase A-DyLight594 (red, middle panel) fixed 15 min post infection. The degree of co-internalization (Merge, right panel) was measured for 10 random cells (R = 0.86 +/- 0.09 (SD). Nuclei were stained with Hoechst (blue). Scale bar is 5 μm. B) Plaque assay of ERAV in the presence of 0–1 mg/ml RNase A. Plaque forming units (pfu) were expressed as percentage of no RNase A control. Data are from three independent experiments with error bars showing standard error.

Techniques Used: Infection, Staining, Plaque Assay

Receptor-decorated liposomes containing fluorescent dye detect PV RNA release. A) Representative images of YoPro-1 encapsulating receptor-decorated liposomes (YRDLs) complexed with PV in the presence or absence of RNase A (50 μg/ml). Note that RNase A was added to the extra-liposomal space after PV-YRDL complexes were formed, but prior to heating the samples for 20 min at 37°C. Images were collected at room temperature using a 20X objective as described in Materials and Methods. Scale bars are 200 μm. B) Normalized histograms showing the number of pixels (y-axis) with a given level of fluorescence (in arbitrary units) (x-axis) of PV-YRDL complexes shown in A in the absence (green curve) and presence (black curve) of RNase A. ( C and D) Representative images of PV RNA (in the absence of liposomes) in the presence of YoPro-1 dye following induction of uncoating by sPVR at 37°C (C) or by heating at 52°C (D) for 20 min in the presence or absence of RNase A (50 μg/ml). Images were collected using a 100X objective as described in Materials and Methods. Scale bars are 40 μm. E) Representative still frames from a time lapse of PV-YRDLs gradually heated from room temperature to 42°C. Average time lapse for averaged image is indicated. After 15 min of imaging a single field of view, a second region of interest was imaged in order to evaluate the influence of photobleaching on the fluorescence intensity (second ROI at 20 min shown on the right-side panel). Images were captured at 100x magnification using a custom built Total Internal Reflectance Fluorescence Microscopy (TIR-FM) setup, attached to an Olympus IX-71 microscope, as described in Material and Methods. Scale bars are 5 μm. F) PV-YRDLs integrated fluorescence intensity obtained as indicated in Materials and Methods (expressed as fold change of T = 1 min, left y-axis) during a 20 min time course (time in min along the x-axis) when the sample was heated from room temperature to 42°C. The temperature of the lens (right y-axis) is shown as a function of time (grey dashed line). Because the objective lens and the sample are 1.18 mm apart (with oil connecting the lens to the sample slide), the temperature of the lens is used to estimate the temperature of the sample. 42°C is the upper limit of the imaging apparatus. The black triangle shows the integrated fluorescence intensity of a region of interest that was imaged at a single time point of 20 min in order to assess photobleaching. G) Representative images of YoPro-1 encapsulating RDL using the same microscope setup described for E. YRDLs were incubated at 37°C for 10 min, alone with no PV (left), or were pre-incubated with PV at room temperature for 10 min to allow complex formation, and then incubated 37°C for 10 min (right). Scale bars are 5 μm.
Figure Legend Snippet: Receptor-decorated liposomes containing fluorescent dye detect PV RNA release. A) Representative images of YoPro-1 encapsulating receptor-decorated liposomes (YRDLs) complexed with PV in the presence or absence of RNase A (50 μg/ml). Note that RNase A was added to the extra-liposomal space after PV-YRDL complexes were formed, but prior to heating the samples for 20 min at 37°C. Images were collected at room temperature using a 20X objective as described in Materials and Methods. Scale bars are 200 μm. B) Normalized histograms showing the number of pixels (y-axis) with a given level of fluorescence (in arbitrary units) (x-axis) of PV-YRDL complexes shown in A in the absence (green curve) and presence (black curve) of RNase A. ( C and D) Representative images of PV RNA (in the absence of liposomes) in the presence of YoPro-1 dye following induction of uncoating by sPVR at 37°C (C) or by heating at 52°C (D) for 20 min in the presence or absence of RNase A (50 μg/ml). Images were collected using a 100X objective as described in Materials and Methods. Scale bars are 40 μm. E) Representative still frames from a time lapse of PV-YRDLs gradually heated from room temperature to 42°C. Average time lapse for averaged image is indicated. After 15 min of imaging a single field of view, a second region of interest was imaged in order to evaluate the influence of photobleaching on the fluorescence intensity (second ROI at 20 min shown on the right-side panel). Images were captured at 100x magnification using a custom built Total Internal Reflectance Fluorescence Microscopy (TIR-FM) setup, attached to an Olympus IX-71 microscope, as described in Material and Methods. Scale bars are 5 μm. F) PV-YRDLs integrated fluorescence intensity obtained as indicated in Materials and Methods (expressed as fold change of T = 1 min, left y-axis) during a 20 min time course (time in min along the x-axis) when the sample was heated from room temperature to 42°C. The temperature of the lens (right y-axis) is shown as a function of time (grey dashed line). Because the objective lens and the sample are 1.18 mm apart (with oil connecting the lens to the sample slide), the temperature of the lens is used to estimate the temperature of the sample. 42°C is the upper limit of the imaging apparatus. The black triangle shows the integrated fluorescence intensity of a region of interest that was imaged at a single time point of 20 min in order to assess photobleaching. G) Representative images of YoPro-1 encapsulating RDL using the same microscope setup described for E. YRDLs were incubated at 37°C for 10 min, alone with no PV (left), or were pre-incubated with PV at room temperature for 10 min to allow complex formation, and then incubated 37°C for 10 min (right). Scale bars are 5 μm.

Techniques Used: Fluorescence, Imaging, Microscopy, Incubation

PV infectivity and RNA integrity are not affected by the presence of high levels of RNase A during the infection process. A) Representative image of HeLa Ohio cells infected with PV-Cy2 (green) in the presence of Dextrans-10 kDa conjugated to Alexa-594 (red) fixed 15 min post-infection. The degree of co-internalization (right-hand side panel) was measured on 10 random cells, R = 0.89 +/- 0.09 (SD). Scale bar is 5 μm. B) Plaque assay of PV in the presence of 0–1 mg/ml RNase A. Plaque forming units were expressed as percentage of no RNase A control. C) Scintillation counting of internalized vs unattached 3 H-U-PV in HeLa Ohio cells in the presence of RNase A (1 mg/ml) or PBS carrier control. D) Scintillation counting of recovered and flow-through samples after a column-based RNA purification procedure of 3 H-U-PV RNA internalized into HeLa Ohio cells in the presence of RNase A (1 mg/ml) or PBS carrier control. E) Scintillation counting of sucrose density gradient (15–30% sucrose, 0.1% SDS, 0.1 M Na acetate. Fraction 1 = top, 15% sucrose) of 3 H-U-PV RNA recovered from HeLa Ohio cells 30 min post-infection in the presence or absence of 1 mg/ml RNase A (PV+HeLa+A, red line, and PV+HeLa, blue line, respectively). Data is expressed as percentage of the total counts per minutes (cpm) loaded onto the gradient. All data are from three independent experiments and error bars show standard error.
Figure Legend Snippet: PV infectivity and RNA integrity are not affected by the presence of high levels of RNase A during the infection process. A) Representative image of HeLa Ohio cells infected with PV-Cy2 (green) in the presence of Dextrans-10 kDa conjugated to Alexa-594 (red) fixed 15 min post-infection. The degree of co-internalization (right-hand side panel) was measured on 10 random cells, R = 0.89 +/- 0.09 (SD). Scale bar is 5 μm. B) Plaque assay of PV in the presence of 0–1 mg/ml RNase A. Plaque forming units were expressed as percentage of no RNase A control. C) Scintillation counting of internalized vs unattached 3 H-U-PV in HeLa Ohio cells in the presence of RNase A (1 mg/ml) or PBS carrier control. D) Scintillation counting of recovered and flow-through samples after a column-based RNA purification procedure of 3 H-U-PV RNA internalized into HeLa Ohio cells in the presence of RNase A (1 mg/ml) or PBS carrier control. E) Scintillation counting of sucrose density gradient (15–30% sucrose, 0.1% SDS, 0.1 M Na acetate. Fraction 1 = top, 15% sucrose) of 3 H-U-PV RNA recovered from HeLa Ohio cells 30 min post-infection in the presence or absence of 1 mg/ml RNase A (PV+HeLa+A, red line, and PV+HeLa, blue line, respectively). Data is expressed as percentage of the total counts per minutes (cpm) loaded onto the gradient. All data are from three independent experiments and error bars show standard error.

Techniques Used: Infection, Plaque Assay, Flow Cytometry, Purification

13) Product Images from "A Hinge Region Cis-proline in Ribonuclease A Acts as a Conformational Gatekeeper for C-terminal Domain Swapping"

Article Title: A Hinge Region Cis-proline in Ribonuclease A Acts as a Conformational Gatekeeper for C-terminal Domain Swapping

Journal: Journal of molecular biology

doi: 10.1016/j.jmb.2010.05.017

HPLC analytical size exclusion chromatography detects dimer in human pancreatic ribonuclease (RNase 1) variants These chromatograms are representative of the results achieved when the RNase 1 variants studied were incubated in 100 mM Tris pH 8.0 at approximately 10 mg/mL total protein for up to 30 days at 4°C or several days at 25°C or 37°C. The R1sP114G mutant shows a dimer peak that elutes from the column ~1.1 minutes before the monomer, while constructs that contain P114 have only a monomeric peak.
Figure Legend Snippet: HPLC analytical size exclusion chromatography detects dimer in human pancreatic ribonuclease (RNase 1) variants These chromatograms are representative of the results achieved when the RNase 1 variants studied were incubated in 100 mM Tris pH 8.0 at approximately 10 mg/mL total protein for up to 30 days at 4°C or several days at 25°C or 37°C. The R1sP114G mutant shows a dimer peak that elutes from the column ~1.1 minutes before the monomer, while constructs that contain P114 have only a monomeric peak.

Techniques Used: High Performance Liquid Chromatography, Size-exclusion Chromatography, Incubation, Mutagenesis, Construct

Determination of dissociation constant for RNase A P114G dimers RNase A P114G was incubated at various concentrations in 100 mM Tris pH 8.0 and allowed to equilibrate for several days. The slope of the linear fit is equal to the dissociation constant (Kd) for dimer formation, 3.66 mM. R 2 = 0.9997.
Figure Legend Snippet: Determination of dissociation constant for RNase A P114G dimers RNase A P114G was incubated at various concentrations in 100 mM Tris pH 8.0 and allowed to equilibrate for several days. The slope of the linear fit is equal to the dissociation constant (Kd) for dimer formation, 3.66 mM. R 2 = 0.9997.

Techniques Used: Incubation

Dimer association and dissociation a) The dimer populations of two samples of RNase A P114G* at 5 mg/mL were monitored as a function of time at 37°C. The dimer association sample started as pure monomer, while the dissociation sample was diluted 1:2 from a 10 mg/mL sample that had equilibrated to 20% dimer. This sample then relaxes to the same dimer population as the association sample as some of the dimer population dissociates over time. b) The WT* (H119A) dimer was isolated and concentrated to a concentration of 27.6 uM (0.751 mg/mL total protein). The sample was then incubated at 37°C and dimer dissociation was allowed to proceed. A population of 14.5 % dimer remained even after a month-long incubation period.
Figure Legend Snippet: Dimer association and dissociation a) The dimer populations of two samples of RNase A P114G* at 5 mg/mL were monitored as a function of time at 37°C. The dimer association sample started as pure monomer, while the dissociation sample was diluted 1:2 from a 10 mg/mL sample that had equilibrated to 20% dimer. This sample then relaxes to the same dimer population as the association sample as some of the dimer population dissociates over time. b) The WT* (H119A) dimer was isolated and concentrated to a concentration of 27.6 uM (0.751 mg/mL total protein). The sample was then incubated at 37°C and dimer dissociation was allowed to proceed. A population of 14.5 % dimer remained even after a month-long incubation period.

Techniques Used: Isolation, Concentration Assay, Incubation

Domain swapping between two inactive mutants of RNase A restores the RNase A active site The two active site residues of RNase A are His 119 (dark blue) and His 12 (red). If one of these residues is mutated, the resulting monomer is inactive. However, upon domain swapping, if either the C terminal (shown) or N-terminal arm is exchanged, the resulting dimer consists of one subunit that contains both active site histidines, and one subunit that contains none. Monomeric structure PDB ID 1RTB, dimeric structure PDB ID 1F0V.
Figure Legend Snippet: Domain swapping between two inactive mutants of RNase A restores the RNase A active site The two active site residues of RNase A are His 119 (dark blue) and His 12 (red). If one of these residues is mutated, the resulting monomer is inactive. However, upon domain swapping, if either the C terminal (shown) or N-terminal arm is exchanged, the resulting dimer consists of one subunit that contains both active site histidines, and one subunit that contains none. Monomeric structure PDB ID 1RTB, dimeric structure PDB ID 1F0V.

Techniques Used:

Structures of the monomer and C-terminal domain-swapped dimer of Ribonuclease A a) PDB ID 1RTB. In monomeric RNase A, Pro 114 adopts a cis conformation b) PDB ID 1F0V. In the C-terminal domain-swapped dimer of RNase A, Pro 114 adopts a trans conformation, resulting in the extension of the hinge between the protein core and the exchanged C-terminal arm. C) PDB ID 1KH8. In the P114G variant of RNase A, the backbone adopts a trans conformation.
Figure Legend Snippet: Structures of the monomer and C-terminal domain-swapped dimer of Ribonuclease A a) PDB ID 1RTB. In monomeric RNase A, Pro 114 adopts a cis conformation b) PDB ID 1F0V. In the C-terminal domain-swapped dimer of RNase A, Pro 114 adopts a trans conformation, resulting in the extension of the hinge between the protein core and the exchanged C-terminal arm. C) PDB ID 1KH8. In the P114G variant of RNase A, the backbone adopts a trans conformation.

Techniques Used: Variant Assay

14) Product Images from "Conserved amino acid networks modulate discrete functional properties in an enzyme superfamily"

Article Title: Conserved amino acid networks modulate discrete functional properties in an enzyme superfamily

Journal: Scientific Reports

doi: 10.1038/s41598-017-03298-4

Structural units tuning conformational dynamics. ( a ) Residues of IC4 mapped on the 3D structure of RNase A (PDB 7RSA). ( b ) Millisecond timescale dynamics of free forms of bovine (bt) RNase A (PDB 7RSA), human (hs) RNases 2 (PDB 1GQV), 3 (PDB 1QMT) and 4 (PDB 1RNF), probed using NMR 15 N-CPMG relaxation dispersion experiments at 500 MHz and 800 MHz and 298 K. Residues showing 15 N-CPMG dispersion profiles with Δ R 2 (1/τ cp ) > 1.5 s −1 are displayed using the space filling representation. Beige color represents residues of loop 4.
Figure Legend Snippet: Structural units tuning conformational dynamics. ( a ) Residues of IC4 mapped on the 3D structure of RNase A (PDB 7RSA). ( b ) Millisecond timescale dynamics of free forms of bovine (bt) RNase A (PDB 7RSA), human (hs) RNases 2 (PDB 1GQV), 3 (PDB 1QMT) and 4 (PDB 1RNF), probed using NMR 15 N-CPMG relaxation dispersion experiments at 500 MHz and 800 MHz and 298 K. Residues showing 15 N-CPMG dispersion profiles with Δ R 2 (1/τ cp ) > 1.5 s −1 are displayed using the space filling representation. Beige color represents residues of loop 4.

Techniques Used: Nuclear Magnetic Resonance

Sector definition for the ptRNase superfamily. ( a ) IC-based sub-matrix of the  C i,j  coupling matrix displaying the top five ICs, resulting in the definition of two sectors – sector 1 corresponding to IC1 and sector 2, comprised of ICs 2, 3, 4 and 5. Color scheme of the diagonal elements in the matrix correspond to the intrinsic conservation of residues, with red and blue colors corresponding to high and low conservation, respectively. Colors of the off-diagonal elements reflects the correlation between residues with the red end of the spectrum corresponding to strongly correlated residue pairs while the blue end of the spectrum indicates uncorrelated interactions. ( b ) Two sectors defined based on IC grouping shown in a. ( c ) Effects of amino acid mutations in sectors 1 (red circles) and 2 (squares) on the catalytic rate ( k cat ) relative to wild type and change in thermal stability (Δ T m  =  T m(mutant)  −  T m(WT) ) in bovine RNase A. The colors of the squares correspond to the IC subgroups defined in Fig.   1 . Mutational data were obtained from the literature and are presented for positions where biochemical properties were characterized under the same conditions using polyC as substrate (residues in bold in Table   S4 ). Wild-type data is shown as a black triangle while non-sector residues are displayed as grey triangles.
Figure Legend Snippet: Sector definition for the ptRNase superfamily. ( a ) IC-based sub-matrix of the C i,j coupling matrix displaying the top five ICs, resulting in the definition of two sectors – sector 1 corresponding to IC1 and sector 2, comprised of ICs 2, 3, 4 and 5. Color scheme of the diagonal elements in the matrix correspond to the intrinsic conservation of residues, with red and blue colors corresponding to high and low conservation, respectively. Colors of the off-diagonal elements reflects the correlation between residues with the red end of the spectrum corresponding to strongly correlated residue pairs while the blue end of the spectrum indicates uncorrelated interactions. ( b ) Two sectors defined based on IC grouping shown in a. ( c ) Effects of amino acid mutations in sectors 1 (red circles) and 2 (squares) on the catalytic rate ( k cat ) relative to wild type and change in thermal stability (Δ T m  =  T m(mutant)  −  T m(WT) ) in bovine RNase A. The colors of the squares correspond to the IC subgroups defined in Fig.  1 . Mutational data were obtained from the literature and are presented for positions where biochemical properties were characterized under the same conditions using polyC as substrate (residues in bold in Table  S4 ). Wild-type data is shown as a black triangle while non-sector residues are displayed as grey triangles.

Techniques Used:

Co-evolving residue networks in the ptRNase superfamily. ( a ) Residue positions of the top five significant eigenmodes (ICs 1–5) are colored red, purple, blue, cyan and teal, respectively, along the primary structure of RNase A. ( b ) Amino acid residues of ICs 1–5, displayed using the space-filling model, mapped on the 3D structure of RNase A (PDB 7RSA). ( c–e ) Each panel shows the scatterplot of sequences (each circle representing a single sequence) along each IC corresponding to sequence variations of positions contributing to each IC. Stacked histograms show the distribution of sequences along each IC. Sequences are colored based on the RNase subtypes found in the entire vertebrate family (RNases 1–8 and inactive members). Sequence distributions along ICs 1 and 2, 3 and 4, 5 and 2 are shown in panels c, d, and e, respectively.
Figure Legend Snippet: Co-evolving residue networks in the ptRNase superfamily. ( a ) Residue positions of the top five significant eigenmodes (ICs 1–5) are colored red, purple, blue, cyan and teal, respectively, along the primary structure of RNase A. ( b ) Amino acid residues of ICs 1–5, displayed using the space-filling model, mapped on the 3D structure of RNase A (PDB 7RSA). ( c–e ) Each panel shows the scatterplot of sequences (each circle representing a single sequence) along each IC corresponding to sequence variations of positions contributing to each IC. Stacked histograms show the distribution of sequences along each IC. Sequences are colored based on the RNase subtypes found in the entire vertebrate family (RNases 1–8 and inactive members). Sequence distributions along ICs 1 and 2, 3 and 4, 5 and 2 are shown in panels c, d, and e, respectively.

Techniques Used: Sequencing

Functional role of co-evolving amino acid networks of ptRNase sectors. ( a ) Amino acid residues of ICs 1–5, displayed as spheres corresponding to Cα atoms, mapped on the 3D structure of RNase A (PDB 7RSA). ICs 1–5 are displayed in red, purple, blue, cyan, and teal spheres, respectively. ( b ) Spheres represent Cα atoms of residues that show NMR chemical shift variations (Δδ) > 0.1 ppm upon incremental titration of RNase A with 5′-AMP (green) and 3′-UMP (marine blue). Brown spheres correspond to residues perturbed by both ligands. Positions of single nucleotide ligands adenosine-5′-monophosphate (5′-AMP) and uridine-3′-monophosphate (3′-UMP), obtained from PDBs 1Z6S and 1O0N, are displayed using stick representations in all Figures. Ligand atoms are colored using the standard coloring scheme – nitrogen, oxygen, carbon and phosphorus as blue, red, white and orange, respectively.
Figure Legend Snippet: Functional role of co-evolving amino acid networks of ptRNase sectors. ( a ) Amino acid residues of ICs 1–5, displayed as spheres corresponding to Cα atoms, mapped on the 3D structure of RNase A (PDB 7RSA). ICs 1–5 are displayed in red, purple, blue, cyan, and teal spheres, respectively. ( b ) Spheres represent Cα atoms of residues that show NMR chemical shift variations (Δδ) > 0.1 ppm upon incremental titration of RNase A with 5′-AMP (green) and 3′-UMP (marine blue). Brown spheres correspond to residues perturbed by both ligands. Positions of single nucleotide ligands adenosine-5′-monophosphate (5′-AMP) and uridine-3′-monophosphate (3′-UMP), obtained from PDBs 1Z6S and 1O0N, are displayed using stick representations in all Figures. Ligand atoms are colored using the standard coloring scheme – nitrogen, oxygen, carbon and phosphorus as blue, red, white and orange, respectively.

Techniques Used: Functional Assay, Nuclear Magnetic Resonance, Titration

15) Product Images from "RNA-Dependent DNA Binding Activity of the Pur Factor, Potentially Involved in DNA Replication and Gene Transcription"

Article Title: RNA-Dependent DNA Binding Activity of the Pur Factor, Potentially Involved in DNA Replication and Gene Transcription

Journal: Gene Expression

doi:

Reconstitution of the PUR binding activity by RNA complementation. The PUR binding activity of fraction 14 of nuclear extracts was examined by mobility shift assay using a 32 P-labeled PUR oligonucleotide. An RNase A-treated fraction 14 was prepared by incubation with RNase A-coupled agarose beads in an aliquot of fraction 14 and subsequent removal of agarose beads by centrifugation (lane 3). RNA was prepared by organic extraction of an other aliquot of the same fraction 14. The complete removal of RNase A was tested by the PUR binding activity of a mixture of RNase A-treated and untreated fractions (lane 4). An excess of RNA corresponding to 20 equivalents of the RNase A-treated fraction was used to reconstitute the Pur factor binding activity (lane 5).
Figure Legend Snippet: Reconstitution of the PUR binding activity by RNA complementation. The PUR binding activity of fraction 14 of nuclear extracts was examined by mobility shift assay using a 32 P-labeled PUR oligonucleotide. An RNase A-treated fraction 14 was prepared by incubation with RNase A-coupled agarose beads in an aliquot of fraction 14 and subsequent removal of agarose beads by centrifugation (lane 3). RNA was prepared by organic extraction of an other aliquot of the same fraction 14. The complete removal of RNase A was tested by the PUR binding activity of a mixture of RNase A-treated and untreated fractions (lane 4). An excess of RNA corresponding to 20 equivalents of the RNase A-treated fraction was used to reconstitute the Pur factor binding activity (lane 5).

Techniques Used: Binding Assay, Activity Assay, Mobility Shift, Labeling, Incubation, Centrifugation

Reconstitution of the PUR binding activity of a RNA-depleted Pur extract with bacterial RNAs. When added to the RNase A-treated fraction 14 of nuclear extracts devoid of PUR binding activity (lane 1), the tRNA crude fraction of  E. coli  (lane 3) is as efficient as RNAs extracted from fraction 14 (lane 2) for restoring the PUR binding activity. Free bacterial RNAs alone fail to form complexes (lane 4). The position of the PUR complex is identified by an arrow.
Figure Legend Snippet: Reconstitution of the PUR binding activity of a RNA-depleted Pur extract with bacterial RNAs. When added to the RNase A-treated fraction 14 of nuclear extracts devoid of PUR binding activity (lane 1), the tRNA crude fraction of E. coli (lane 3) is as efficient as RNAs extracted from fraction 14 (lane 2) for restoring the PUR binding activity. Free bacterial RNAs alone fail to form complexes (lane 4). The position of the PUR complex is identified by an arrow.

Techniques Used: Binding Assay, Activity Assay

RNAs interacting with Pur are present in rabbit retic-ulocyte lysates. (A) Sensitivity to RNase A of the PUR binding activity of the in vitro translated Purα protein. The ability of an in vitro-translated Purα protein to bind 32 P-labeled PUR oligonucleotides was examined by mobility shift assay. Binding reactions were done with the reticulocyte lysate immediately after the translation reaction (lane 1), or after an additional 30-min incubation at 37°C in the absence (lane 2) or presence (lane 3) of RNase A. (B) Presence in the rabbit reticulocyte lysate of RNAs similar to the Pur-associated RNAs of fraction 14 of RSV-infected QEF nuclear extracts. Total RNAs were prepared from fraction 14 of nuclear extracts from RSV-infected QEF (lane 1), or the rabbit reticulocyte lysate (lane 2), and 3′ end labeled using the T4 RNA polymerase, and run in parallel in a denaturing 20% polyacrylamide gel, along with 22, 27, and 32 mer radioactive oligonucleotides as molecular weight markers. Identical 28- and 29-bases-long RNAs, similar to those previously found associated with the PUR complexes, are present in both preparations, and are indicated by arrows.
Figure Legend Snippet: RNAs interacting with Pur are present in rabbit retic-ulocyte lysates. (A) Sensitivity to RNase A of the PUR binding activity of the in vitro translated Purα protein. The ability of an in vitro-translated Purα protein to bind 32 P-labeled PUR oligonucleotides was examined by mobility shift assay. Binding reactions were done with the reticulocyte lysate immediately after the translation reaction (lane 1), or after an additional 30-min incubation at 37°C in the absence (lane 2) or presence (lane 3) of RNase A. (B) Presence in the rabbit reticulocyte lysate of RNAs similar to the Pur-associated RNAs of fraction 14 of RSV-infected QEF nuclear extracts. Total RNAs were prepared from fraction 14 of nuclear extracts from RSV-infected QEF (lane 1), or the rabbit reticulocyte lysate (lane 2), and 3′ end labeled using the T4 RNA polymerase, and run in parallel in a denaturing 20% polyacrylamide gel, along with 22, 27, and 32 mer radioactive oligonucleotides as molecular weight markers. Identical 28- and 29-bases-long RNAs, similar to those previously found associated with the PUR complexes, are present in both preparations, and are indicated by arrows.

Techniques Used: Binding Assay, Activity Assay, In Vitro, Labeling, Mobility Shift, Incubation, Infection, Molecular Weight

Proteinase K and RNase A sensitivity of the PUR complex. Mobility shift assays were carried out using the PUR probe and nuclear extracts from RSV-infected QEF, either untreated (lanes 1, 13), or pretreated with proteinase K (lane 3) or RNase A (lanes 6–9). Nuclear extracts were also preincubated in the same conditions in the presence of enzymatically inactive forms of proteinase K (K 0 , lane 4) and RNase A (A 0 , lane 12). The RNase inhibitor (RNasin) was added (lane 9) prior to incubation with RNase A.
Figure Legend Snippet: Proteinase K and RNase A sensitivity of the PUR complex. Mobility shift assays were carried out using the PUR probe and nuclear extracts from RSV-infected QEF, either untreated (lanes 1, 13), or pretreated with proteinase K (lane 3) or RNase A (lanes 6–9). Nuclear extracts were also preincubated in the same conditions in the presence of enzymatically inactive forms of proteinase K (K 0 , lane 4) and RNase A (A 0 , lane 12). The RNase inhibitor (RNasin) was added (lane 9) prior to incubation with RNase A.

Techniques Used: Mobility Shift, Infection, Incubation

16) Product Images from "Optimization of ribosome profiling using low-input brain tissue from fragile X syndrome model mice"

Article Title: Optimization of ribosome profiling using low-input brain tissue from fragile X syndrome model mice

Journal: Nucleic Acids Research

doi: 10.1093/nar/gky1292

RPF GC content is RNase-species independent. ( A ) 3.8 A 260 homogenate from hippocampi of one P35 male mouse was digested with 100ng RNase A (Sigma, # R4875) + 60U RNase T1 (Thermo Fisher Scientific, #EN0542)/ A 260 , at 25°C for 30min and applied to a 10–50% (w/v) sucrose gradient. ( B ) 3.8 A 260 homogenate from hippocampi of one P35 mouse was digested with 5U RNase I (Ambion, #AM2294)/ A 260 , at 25°C for 45min and applied to a 10–50% (w/v) sucrose gradient. ( C ) Nucleotide composition at each position of RPFs mapped to CDS from ribosomes in (A). ( D ) Nucleotide composition at each position of RPFs mapped to CDS from ribosomes in (B). ( E ) Nucleotide composition at each position of RPFs mapped to CDS from mouse embryonic stem cells (mESCs) (data from Ingolia et al. ) ( 16 ). A 600 μl aliquot of lysate was treated with 15 μl RNase I at 100 U/μl for 45 min at 25°C. ( F ) Nucleotide composition at each position of RPFs mapped to CDS from human embryonic stem cell (hESC)-derived neurons (data from Grabole et al. ) ( 42 ). 5 U TruSeq Ribo Profile Nuclease/ A 260 at 25°C for 45 min.
Figure Legend Snippet: RPF GC content is RNase-species independent. ( A ) 3.8 A 260 homogenate from hippocampi of one P35 male mouse was digested with 100ng RNase A (Sigma, # R4875) + 60U RNase T1 (Thermo Fisher Scientific, #EN0542)/ A 260 , at 25°C for 30min and applied to a 10–50% (w/v) sucrose gradient. ( B ) 3.8 A 260 homogenate from hippocampi of one P35 mouse was digested with 5U RNase I (Ambion, #AM2294)/ A 260 , at 25°C for 45min and applied to a 10–50% (w/v) sucrose gradient. ( C ) Nucleotide composition at each position of RPFs mapped to CDS from ribosomes in (A). ( D ) Nucleotide composition at each position of RPFs mapped to CDS from ribosomes in (B). ( E ) Nucleotide composition at each position of RPFs mapped to CDS from mouse embryonic stem cells (mESCs) (data from Ingolia et al. ) ( 16 ). A 600 μl aliquot of lysate was treated with 15 μl RNase I at 100 U/μl for 45 min at 25°C. ( F ) Nucleotide composition at each position of RPFs mapped to CDS from human embryonic stem cell (hESC)-derived neurons (data from Grabole et al. ) ( 42 ). 5 U TruSeq Ribo Profile Nuclease/ A 260 at 25°C for 45 min.

Techniques Used: Derivative Assay

RPF GC content and length depend on the RNase digestion protocol. ( A ) Lysates from human iPSC neuron samples spanning a wide range of amounts were digested with 100 ng RNase A + 60U RNase T1/ A 260 at 25°C for 30 min. Monosomal RNA was extracted from monosomal fractions of sucrose gradients and quantified with Nanodrop. GC contents were calculated as in Figure 2A and the peaks of length distributions of RPFs mapped to CDS were also determined. Scatter plots with Pearson correlation coefficients show the negative correlation between 80S monosomal RNA amounts (log 2 scale) and the GC contents (black) or RPF lengths (red). ( B ) Lysates from human iPSC samples were digested with 20 ng RNase A + 12 U RNase T1/ A 260 at 25°C for 30 min. Scatter plots with Pearson correlation coefficients show the negative correlation between 80S monosomal RNA amounts (log 2 scale) and the GC contents (black) or RPF lengths (red). ( C ) Nucleotide composition at each position of RPFs mapped to CDS from mESC-derived neurons with an alternative protocol of RNase digestion (data from Zappulo et al. ) ( 43 ). 70 U RNase I at 25°C for 40 min.
Figure Legend Snippet: RPF GC content and length depend on the RNase digestion protocol. ( A ) Lysates from human iPSC neuron samples spanning a wide range of amounts were digested with 100 ng RNase A + 60U RNase T1/ A 260 at 25°C for 30 min. Monosomal RNA was extracted from monosomal fractions of sucrose gradients and quantified with Nanodrop. GC contents were calculated as in Figure 2A and the peaks of length distributions of RPFs mapped to CDS were also determined. Scatter plots with Pearson correlation coefficients show the negative correlation between 80S monosomal RNA amounts (log 2 scale) and the GC contents (black) or RPF lengths (red). ( B ) Lysates from human iPSC samples were digested with 20 ng RNase A + 12 U RNase T1/ A 260 at 25°C for 30 min. Scatter plots with Pearson correlation coefficients show the negative correlation between 80S monosomal RNA amounts (log 2 scale) and the GC contents (black) or RPF lengths (red). ( C ) Nucleotide composition at each position of RPFs mapped to CDS from mESC-derived neurons with an alternative protocol of RNase digestion (data from Zappulo et al. ) ( 43 ). 70 U RNase I at 25°C for 40 min.

Techniques Used: Derivative Assay

The GC-content correlated batch effects are caused by incomplete RNase digestion. ( A ) Hippocampi from one P35 WT mouse were homogenized and the homogenate was aliquoted for the titration experiment. 0.5 unit A 260 homogenate containing 2 μg RNA (measured with Qubit HS RNA kit) in 0.3 ml volume was used for digestion at each RNase concentration. Digested homogenates were separated on 10–50% (w/v) sucrose gradients. Profile of hippocampal ribosomes after the digestion at the lowest concentration1 [Conc.1, 4.8ng RNase A (Ambion, #AM2270) + 0.6 U RNase T1 (Thermo Fisher Scientific, #EN0542)/μg RNA × 2 μg RNA in 0.3 ml at 25°C for 30 min] and sucrose gradient fractionation. ( B ) Profile of hippocampal ribosomes after the digestion at the concentration2 (Conc.2, 24 ng RNase A + 3U RNase T1/μg RNA × 2 μg RNA in 0.3 ml at 25°C for 30 min) and sucrose gradient fractionation. ( C ) Profile of hippocampal ribosomes after the digestion at the concentration3 (Conc.3, 120 ng RNase A + 15 U RNase T1/μg RNA × 2 μg RNA in 0.3 ml at 25°C for 30 min) and sucrose gradient fractionation. ( D ) Profile of hippocampal ribosomes after the digestion at the concentration4 (Conc.4, 600 ng RNase A + 75 U RNase T1/μg RNA × 2 μg RNA in 0.3 ml at 25°C for 30 min) and sucrose gradient fractionation. ( E ) Profile of hippocampal ribosomes after the digestion at the highest concentration5 (Conc.5, 3000 ng RNase A + 375 U RNase T1/μg RNA × 2 μg RNA RNA in 0.3 ml at 25°C for 30 min) and sucrose gradient fractionation. ( F ) Scatter plots with Pearson correlation coefficients show the negative correlation between RNase concentrations (log 5 scale) and the GC contents (black) or RPF lengths (red).
Figure Legend Snippet: The GC-content correlated batch effects are caused by incomplete RNase digestion. ( A ) Hippocampi from one P35 WT mouse were homogenized and the homogenate was aliquoted for the titration experiment. 0.5 unit A 260 homogenate containing 2 μg RNA (measured with Qubit HS RNA kit) in 0.3 ml volume was used for digestion at each RNase concentration. Digested homogenates were separated on 10–50% (w/v) sucrose gradients. Profile of hippocampal ribosomes after the digestion at the lowest concentration1 [Conc.1, 4.8ng RNase A (Ambion, #AM2270) + 0.6 U RNase T1 (Thermo Fisher Scientific, #EN0542)/μg RNA × 2 μg RNA in 0.3 ml at 25°C for 30 min] and sucrose gradient fractionation. ( B ) Profile of hippocampal ribosomes after the digestion at the concentration2 (Conc.2, 24 ng RNase A + 3U RNase T1/μg RNA × 2 μg RNA in 0.3 ml at 25°C for 30 min) and sucrose gradient fractionation. ( C ) Profile of hippocampal ribosomes after the digestion at the concentration3 (Conc.3, 120 ng RNase A + 15 U RNase T1/μg RNA × 2 μg RNA in 0.3 ml at 25°C for 30 min) and sucrose gradient fractionation. ( D ) Profile of hippocampal ribosomes after the digestion at the concentration4 (Conc.4, 600 ng RNase A + 75 U RNase T1/μg RNA × 2 μg RNA in 0.3 ml at 25°C for 30 min) and sucrose gradient fractionation. ( E ) Profile of hippocampal ribosomes after the digestion at the highest concentration5 (Conc.5, 3000 ng RNase A + 375 U RNase T1/μg RNA × 2 μg RNA RNA in 0.3 ml at 25°C for 30 min) and sucrose gradient fractionation. ( F ) Scatter plots with Pearson correlation coefficients show the negative correlation between RNase concentrations (log 5 scale) and the GC contents (black) or RPF lengths (red).

Techniques Used: Titration, Concentration Assay, Fractionation

17) Product Images from "Refolding and simultaneous purification by three-phase partitioning of recombinant proteins from inclusion bodies"

Article Title: Refolding and simultaneous purification by three-phase partitioning of recombinant proteins from inclusion bodies

Journal: Protein Science : A Publication of the Protein Society

doi: 10.1110/ps.036939.108

The binding isotherm of RNase A and 3′-CMP. RNase A (55 μM) in 0.2 M sodium acetate buffer, pH 6.0 containing 0.2 M NaCl, was titrated with small injections of 3′-CMP solution in the same buffer. The enthalpy data were fitted to a single site binding model. ( A ) Binding isotherm for refolded RNase A; ( B ) binding isotherm for Sigma RNase A.
Figure Legend Snippet: The binding isotherm of RNase A and 3′-CMP. RNase A (55 μM) in 0.2 M sodium acetate buffer, pH 6.0 containing 0.2 M NaCl, was titrated with small injections of 3′-CMP solution in the same buffer. The enthalpy data were fitted to a single site binding model. ( A ) Binding isotherm for refolded RNase A; ( B ) binding isotherm for Sigma RNase A.

Techniques Used: Binding Assay

Spectroscopic characterization of refolded RNase A. ( A ) Fluorescence emission spectra of RNase A. The samples at a concentration of 100 μg/mL −1 (∼7 μM) were excited at 278 nm and the emission spectra from 290 to 400 nm were recorded, using excitation and emission slit widths of 2 nm and 5 nm, respectively. Fluorescence spectra of unfolded RNase A (3), refolded RNase A (2), Sigma RNase A (1) are shown after correction for buffer contribution. ( B ) The far-UV CD spectra of refolded RNase A (○) and Sigma RNase A (△) for 0.5 mg/mL −1 protein recorded in a 1-mm path length cuvette from 200 to 250 nm. ( C ) The near-UV CD spectra of the two samples recorded for 0.5 mg/mL −1 protein in a 1-cm path length cuvette from 250 to 350 nm. The symbols are the same as in B .
Figure Legend Snippet: Spectroscopic characterization of refolded RNase A. ( A ) Fluorescence emission spectra of RNase A. The samples at a concentration of 100 μg/mL −1 (∼7 μM) were excited at 278 nm and the emission spectra from 290 to 400 nm were recorded, using excitation and emission slit widths of 2 nm and 5 nm, respectively. Fluorescence spectra of unfolded RNase A (3), refolded RNase A (2), Sigma RNase A (1) are shown after correction for buffer contribution. ( B ) The far-UV CD spectra of refolded RNase A (○) and Sigma RNase A (△) for 0.5 mg/mL −1 protein recorded in a 1-mm path length cuvette from 200 to 250 nm. ( C ) The near-UV CD spectra of the two samples recorded for 0.5 mg/mL −1 protein in a 1-cm path length cuvette from 250 to 350 nm. The symbols are the same as in B .

Techniques Used: Fluorescence, Concentration Assay

TPP purification monitored by SDS-PAGE. ( A ) Fifteen percent SDS-PAGE analysis of RNase A inclusion bodies at different steps of TPP; (lane 1 ) unwashed inclusion bodies; (lane 2 ) washed inclusion bodies with 50 mM PBS/pH 7.4/1 mM EDTA containing 2 M urea; (lane 3 ) commercial RNase A (Sigma Chemical Co.); (lane 4 ) refolded RNase A obtained from interfacial precipitate of second TPP; (lane 5 ) interfacial precipitate of first TPP; (lane 6 ) aqueous phase of second TPP. ( B ) Fifteen percent SDS-PAGE analysis of inclusion bodies of CcdB mutants subjected to TPP; (lane 1 ) unwashed inclusion bodies of CcdB-F17P; (lane 2 ) washed inclusion bodies of CcdB-F17P with 50 mM PBS/pH 7.4/0.5% Triton X-100; (lane 3 ) interfacial precipitate of first TPP; (lane 4 ) aqueous phase of second TPP; (lane 5 ) refolded and purified CcdB-F17P by TPP; (lane 6 ) molecular weight marker; (lane 7 ) unwashed inclusion bodies of CcdB-M97K; (lane 8 ) washed inclusion bodies of CcdB-M97K; (lane 9 ) interfacial precipitate of first TPP; (lane 10 ) aqueous phase of second TPP; (lane 11 ) refolded and purified CcdB-M97K by TPP. ( C ) Fifteen percent SDS-PAGE analysis of inclusion bodies of CD4D12 subjected to TPP CD4D12; (lane 1 ) molecular weight marker; (lane 2 ) unwashed inclusion bodies of CD4D12; (lane 3 ) washed inclusion bodies of CD4D12 with 50 mM PBS/pH 7.4/0.5% Triton X-100; (lane 4 ) interfacial precipitate of first TPP; (lane 5 ) aqueous phase of second TPP; (lane 6 ) refolded and purified CD4D12 by TPP; (lane 7 ) refolded and purified CD4D12 after subjected to the 50-kDa polyethersulfone membrane (PALL Lifesciences) once; (lane 8 ) refolded and purified CD4D12 after subjected to the 50-kDa polyethersulfone membrane (PALL Lifesciences) twice; (lane 9 ) refolded and purified CD4D12 in the absence of DTT.
Figure Legend Snippet: TPP purification monitored by SDS-PAGE. ( A ) Fifteen percent SDS-PAGE analysis of RNase A inclusion bodies at different steps of TPP; (lane 1 ) unwashed inclusion bodies; (lane 2 ) washed inclusion bodies with 50 mM PBS/pH 7.4/1 mM EDTA containing 2 M urea; (lane 3 ) commercial RNase A (Sigma Chemical Co.); (lane 4 ) refolded RNase A obtained from interfacial precipitate of second TPP; (lane 5 ) interfacial precipitate of first TPP; (lane 6 ) aqueous phase of second TPP. ( B ) Fifteen percent SDS-PAGE analysis of inclusion bodies of CcdB mutants subjected to TPP; (lane 1 ) unwashed inclusion bodies of CcdB-F17P; (lane 2 ) washed inclusion bodies of CcdB-F17P with 50 mM PBS/pH 7.4/0.5% Triton X-100; (lane 3 ) interfacial precipitate of first TPP; (lane 4 ) aqueous phase of second TPP; (lane 5 ) refolded and purified CcdB-F17P by TPP; (lane 6 ) molecular weight marker; (lane 7 ) unwashed inclusion bodies of CcdB-M97K; (lane 8 ) washed inclusion bodies of CcdB-M97K; (lane 9 ) interfacial precipitate of first TPP; (lane 10 ) aqueous phase of second TPP; (lane 11 ) refolded and purified CcdB-M97K by TPP. ( C ) Fifteen percent SDS-PAGE analysis of inclusion bodies of CD4D12 subjected to TPP CD4D12; (lane 1 ) molecular weight marker; (lane 2 ) unwashed inclusion bodies of CD4D12; (lane 3 ) washed inclusion bodies of CD4D12 with 50 mM PBS/pH 7.4/0.5% Triton X-100; (lane 4 ) interfacial precipitate of first TPP; (lane 5 ) aqueous phase of second TPP; (lane 6 ) refolded and purified CD4D12 by TPP; (lane 7 ) refolded and purified CD4D12 after subjected to the 50-kDa polyethersulfone membrane (PALL Lifesciences) once; (lane 8 ) refolded and purified CD4D12 after subjected to the 50-kDa polyethersulfone membrane (PALL Lifesciences) twice; (lane 9 ) refolded and purified CD4D12 in the absence of DTT.

Techniques Used: Purification, SDS Page, Molecular Weight, Marker

18) Product Images from "Mammalian splicing factor SF1 interacts with SURP domains of U2 snRNP-associated proteins"

Article Title: Mammalian splicing factor SF1 interacts with SURP domains of U2 snRNP-associated proteins

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkv952

Determination of the SURP-ID of SF1. ( A ) Scheme of mutant SF1 proteins. Boxes represent known protein domains: ULM, UHM ligand motif; KH/QUA2, K-homology/Quaking2 domain; Zn, zinc knuckle. Numbering above full-length SF1 refers to amino acids. The names of SF1 mutants are shown on the left; numbers on the right refer to the residues comprising the proteins. ( B ) and ( C ) GST pull-down of mutant SF1 proteins with SURP domains. GST-tagged SF3a120-SURP1, CHERP-SURP, SFSWAP-SURP2, U2AF65-UHM and GST alone, bound to glutathione agarose (as indicated on the right), were incubated with mutant His 6 - (B) or His 6 -MBP-tagged (C) SF1 proteins as indicated above the figures. Bound proteins were separated by 10% SDS-PAGE and western blotted with anti-His 6  (top panels) and anti-GST (bottom panels). The input (10% of the total) is shown at the bottom. ( D ) Co-IP. GFP-tagged SF1-C370 and -C302 were transiently expressed in HeLa cells. Total cell lysates were RNase A-treated and incubated with Dynabeads Protein G-coupled anti-GFP. Input (I; 0.5% of the total) and bound proteins (B) were separated by 7.5% SDS-PAGE and western blotted with anti-SF3a120, anti-U2AF65 and anti-GFP as indicated on the right. The migration of protein markers (in kDa) is shown on the left. ( E ) Multiple sequence alignment of SF1 proteins. SF1 sequences taken from the UniProt database ( www.uniprot.org ) were aligned with ClustalW2 ( www.ebi.ac.uk/Tools/msa/clustalw2/ ). The region from the zinc knuckle of human SF1 and 40 amino acids C-terminal of this domain is shown. Numbering is given for human SF1. Amino acids identical in more than 50% of the sequences are marked.
Figure Legend Snippet: Determination of the SURP-ID of SF1. ( A ) Scheme of mutant SF1 proteins. Boxes represent known protein domains: ULM, UHM ligand motif; KH/QUA2, K-homology/Quaking2 domain; Zn, zinc knuckle. Numbering above full-length SF1 refers to amino acids. The names of SF1 mutants are shown on the left; numbers on the right refer to the residues comprising the proteins. ( B ) and ( C ) GST pull-down of mutant SF1 proteins with SURP domains. GST-tagged SF3a120-SURP1, CHERP-SURP, SFSWAP-SURP2, U2AF65-UHM and GST alone, bound to glutathione agarose (as indicated on the right), were incubated with mutant His 6 - (B) or His 6 -MBP-tagged (C) SF1 proteins as indicated above the figures. Bound proteins were separated by 10% SDS-PAGE and western blotted with anti-His 6 (top panels) and anti-GST (bottom panels). The input (10% of the total) is shown at the bottom. ( D ) Co-IP. GFP-tagged SF1-C370 and -C302 were transiently expressed in HeLa cells. Total cell lysates were RNase A-treated and incubated with Dynabeads Protein G-coupled anti-GFP. Input (I; 0.5% of the total) and bound proteins (B) were separated by 7.5% SDS-PAGE and western blotted with anti-SF3a120, anti-U2AF65 and anti-GFP as indicated on the right. The migration of protein markers (in kDa) is shown on the left. ( E ) Multiple sequence alignment of SF1 proteins. SF1 sequences taken from the UniProt database ( www.uniprot.org ) were aligned with ClustalW2 ( www.ebi.ac.uk/Tools/msa/clustalw2/ ). The region from the zinc knuckle of human SF1 and 40 amino acids C-terminal of this domain is shown. Numbering is given for human SF1. Amino acids identical in more than 50% of the sequences are marked.

Techniques Used: Mutagenesis, Incubation, SDS Page, Western Blot, Co-Immunoprecipitation Assay, Migration, Sequencing

Deletion of the SF1 SURP-ID does not reduce U2AF65 binding to the pre-mRNA. ( A ) and ( B ) U2AF65 UV cross-linking to AdML 3′ splice site substrates. Splicing reactions containing radio-labeled RNA with a consensus (A) or weak (B) BPS, mock or SF1-depleted (ΔSF1) extracts complemented with 2.2 pmole His 6 -SF1-C370 or -C302 as indicated were incubated at 30°C for 15 min. Samples were UV cross-linked, RNase A-treated and immunoprecipitated with control IgG or anti-U2AF65, as indicated. RNA–protein complexes were separated by 10% SDS-PAGE. Gels were dried and exposed to PhosphorImager screens. The top panels show representative results of triplicate experiments; quantifications are shown in the bottom panels. ‘% U2AF65-bound RNA’ indicates the percentage of the intensity of the cross-linked RNAs normalized to the RNA immunoprecipitated with anti-U2AF65 from mock-treated extract. Data are shown as mean value ± SEM.
Figure Legend Snippet: Deletion of the SF1 SURP-ID does not reduce U2AF65 binding to the pre-mRNA. ( A ) and ( B ) U2AF65 UV cross-linking to AdML 3′ splice site substrates. Splicing reactions containing radio-labeled RNA with a consensus (A) or weak (B) BPS, mock or SF1-depleted (ΔSF1) extracts complemented with 2.2 pmole His 6 -SF1-C370 or -C302 as indicated were incubated at 30°C for 15 min. Samples were UV cross-linked, RNase A-treated and immunoprecipitated with control IgG or anti-U2AF65, as indicated. RNA–protein complexes were separated by 10% SDS-PAGE. Gels were dried and exposed to PhosphorImager screens. The top panels show representative results of triplicate experiments; quantifications are shown in the bottom panels. ‘% U2AF65-bound RNA’ indicates the percentage of the intensity of the cross-linked RNAs normalized to the RNA immunoprecipitated with anti-U2AF65 from mock-treated extract. Data are shown as mean value ± SEM.

Techniques Used: Binding Assay, Labeling, Incubation, Immunoprecipitation, SDS Page

Analysis of the interaction of SF1 with SURP domain-containing proteins. ( A ) Scheme of SURP domain-containing proteins identified in Y2H screens. The domain structure of SF3a120, CHERP and SFSWAP is shown with SURP domains indicated in dark grey. Other domains are shown in light grey: CID, RNA polymerase II-binding domain; G-patch, G-patch domain; RS domain, Arg/Ser-rich domain; UBL, ubiquitin-like domain. Numbering was taken from UniProt entries ( www.uniprot.org ). The smallest selected interaction domain (ΣSID) deduced from cDNAs found in the Y2H screens is indicated below the proteins (numbering according to human prote ins). ( B ) Co-IP. HeLa cell nuclear extract was incubated with Dynabeads Protein G coated with anti-SF1 or control IgG. Input (I; 10% of total), bound (B) and unbound (U) fractions were separated by 7.5% SDS-PAGE (10% for anti-H1) followed by western blotting with antibodies against the proteins indicated on the right side of each panel. ( C ) GST pull-down. His 6 -tagged SF1-C370 was incubated with GST alone, GST-tagged U2AF65-UHM, SF3a120-SURP1, CHERP-SURP or SFSWAP-SURP2 bound to glutathione-agarose as indicated above the figure. GST-tagged proteins were mock-treated (−) or digested with RNase A (+) as shown. The His 6 -SF1-C370 input (I; 10% of total) and bound proteins were separated by 10% SDS-PAGE and western blotted with anti-His 6 (top) and anti-GST antibodies (bottom). The migration of protein markers is indicated in kDa on the left.
Figure Legend Snippet: Analysis of the interaction of SF1 with SURP domain-containing proteins. ( A ) Scheme of SURP domain-containing proteins identified in Y2H screens. The domain structure of SF3a120, CHERP and SFSWAP is shown with SURP domains indicated in dark grey. Other domains are shown in light grey: CID, RNA polymerase II-binding domain; G-patch, G-patch domain; RS domain, Arg/Ser-rich domain; UBL, ubiquitin-like domain. Numbering was taken from UniProt entries ( www.uniprot.org ). The smallest selected interaction domain (ΣSID) deduced from cDNAs found in the Y2H screens is indicated below the proteins (numbering according to human prote ins). ( B ) Co-IP. HeLa cell nuclear extract was incubated with Dynabeads Protein G coated with anti-SF1 or control IgG. Input (I; 10% of total), bound (B) and unbound (U) fractions were separated by 7.5% SDS-PAGE (10% for anti-H1) followed by western blotting with antibodies against the proteins indicated on the right side of each panel. ( C ) GST pull-down. His 6 -tagged SF1-C370 was incubated with GST alone, GST-tagged U2AF65-UHM, SF3a120-SURP1, CHERP-SURP or SFSWAP-SURP2 bound to glutathione-agarose as indicated above the figure. GST-tagged proteins were mock-treated (−) or digested with RNase A (+) as shown. The His 6 -SF1-C370 input (I; 10% of total) and bound proteins were separated by 10% SDS-PAGE and western blotted with anti-His 6 (top) and anti-GST antibodies (bottom). The migration of protein markers is indicated in kDa on the left.

Techniques Used: Binding Assay, Co-Immunoprecipitation Assay, Incubation, SDS Page, Western Blot, Migration

19) Product Images from "Noncoding RNA Ginir functions as an oncogene by associating with centrosomal proteins"

Article Title: Noncoding RNA Ginir functions as an oncogene by associating with centrosomal proteins

Journal: PLoS Biology

doi: 10.1371/journal.pbio.2004204

Ginir RNA impairs interaction between Cep112 and Brca1 proteins leading to genomic instability. (A) RNA pull-down with biotinylated Ginir RNA in NIH/3T3 cells followed by western blotting with Brca1 antibody (sc-646, Santa Cruz). Pull-down with unbiotinylated RNA probe served as control for nonspecific binding. (B-D) RIP performed using both Cep112 and Brca1 antibodies followed by RNA isolation and RT-PCR with Ginir specific primers (G2F-G2R) in NIH/3T3-GinirA (B) and NIH/3T3-GinirB (C) cells. RIP assay was also followed by RT-PCR using nonspecific primers for U6 snRNA (D). Anti-IgG IP served as control for nonspecific interaction. (E) In silico model of Cep112 and Brca1 interaction generated through computational docking using ZDOCK tool. (F and G) Co-IP of Cep112 and Brca1 proteins in NIH/3T3-EV cells. IP was performed with Brca1 antibody (sc-646, Santa Cruz) followed by immunoblotting with Cep112 antibody (sc-246162, Santa Cruz) (F) and vice versa (G). Anti-IgG IP served as a control for nonspecific binding to the antibody. (H and I) Western blotting with Cep112 antibody (24928-1-AP, Proteintech) for validation of Flag-Cep112 overexpression in NIH/3T3 cells (H). Fifty μg of whole-cell protein lysates from each of the mentioned cell lines was loaded on 7% SDS-PAGE. Tubulin served as internal loading control (I). (J) Co-IP of Brca1 and Flag-Cep112 in NIH-Flag-Cep112 cells. IP was performed with Flag1 antibody followed by immunoblotting with Brca1 antibody (20649-1-AP, Proteintech). Anti-IgG IP served as a control for nonspecific binding to the antibody. (K) Co-IP of Brca1 and Cep112 proteins in NIH/3T3-EV, NIH/3T3-Ginir, and NIH-Ginir-shGinir2 cells. IP was performed with Brca1 antibody (sc-646, Santa Cruz) followed by immunoblotting with Cep112 antibody (24928-1-AP, Proteintech). Anti-IgG IP served as a control for nonspecific binding to the antibody. (L) Confocal images showing colocalisation of Brca1 with γ-tubulin in NIH/3T3-EV cell line. Nuclei were stained with DAPI. Scale bars, 10 μm. (M) Confocal images showing colocalisation of Brca1 protein with Cep112 protein in NIH/3T3-EV cell line. Nuclei were stained with DAPI. Scale bars, 20 μm. (N) Confocal imaging for Brca1 expression in NIH/3T3-EV and NIH/3T3-Ginir cells. Scale bars, 20 μm. (O) RNA-FISH using Ginir-specific probe (probe 1, FAM labelled) in NIH/3T3-EV cells visualised by confocal imaging. Scale bars, 20 μm. (P) Co-IP of Brca1 and Cep112 in NIH/3T3-Ginir(C) cells wherein lysates were treated independently with RNase (A, H, and III mix) or RNasin. Both RNase- and RNasin-treated lysates were immunoprecipitated with Brca1 antibody (sc-646, Santa Cruz) and blotted using Cep112 antibody (sc-246163, Santa Cruz). IP with anti-IgG served as control. Brca1, breast cancer type 1 susceptibility protein; Cep112, centrosomal protein 112; FAM, fluorescein amidite; Ginir, Genomic Instability Inducing RNA; IgG, immunoglobulin G; IP, immunoprecipitation; RIP, RNA-immunoprecipitation; RNasin, RNase inhibitor; RT-PCR, reverse transcription polymerase chain reaction; snRNA, small nuclear RNA.
Figure Legend Snippet: Ginir RNA impairs interaction between Cep112 and Brca1 proteins leading to genomic instability. (A) RNA pull-down with biotinylated Ginir RNA in NIH/3T3 cells followed by western blotting with Brca1 antibody (sc-646, Santa Cruz). Pull-down with unbiotinylated RNA probe served as control for nonspecific binding. (B-D) RIP performed using both Cep112 and Brca1 antibodies followed by RNA isolation and RT-PCR with Ginir specific primers (G2F-G2R) in NIH/3T3-GinirA (B) and NIH/3T3-GinirB (C) cells. RIP assay was also followed by RT-PCR using nonspecific primers for U6 snRNA (D). Anti-IgG IP served as control for nonspecific interaction. (E) In silico model of Cep112 and Brca1 interaction generated through computational docking using ZDOCK tool. (F and G) Co-IP of Cep112 and Brca1 proteins in NIH/3T3-EV cells. IP was performed with Brca1 antibody (sc-646, Santa Cruz) followed by immunoblotting with Cep112 antibody (sc-246162, Santa Cruz) (F) and vice versa (G). Anti-IgG IP served as a control for nonspecific binding to the antibody. (H and I) Western blotting with Cep112 antibody (24928-1-AP, Proteintech) for validation of Flag-Cep112 overexpression in NIH/3T3 cells (H). Fifty μg of whole-cell protein lysates from each of the mentioned cell lines was loaded on 7% SDS-PAGE. Tubulin served as internal loading control (I). (J) Co-IP of Brca1 and Flag-Cep112 in NIH-Flag-Cep112 cells. IP was performed with Flag1 antibody followed by immunoblotting with Brca1 antibody (20649-1-AP, Proteintech). Anti-IgG IP served as a control for nonspecific binding to the antibody. (K) Co-IP of Brca1 and Cep112 proteins in NIH/3T3-EV, NIH/3T3-Ginir, and NIH-Ginir-shGinir2 cells. IP was performed with Brca1 antibody (sc-646, Santa Cruz) followed by immunoblotting with Cep112 antibody (24928-1-AP, Proteintech). Anti-IgG IP served as a control for nonspecific binding to the antibody. (L) Confocal images showing colocalisation of Brca1 with γ-tubulin in NIH/3T3-EV cell line. Nuclei were stained with DAPI. Scale bars, 10 μm. (M) Confocal images showing colocalisation of Brca1 protein with Cep112 protein in NIH/3T3-EV cell line. Nuclei were stained with DAPI. Scale bars, 20 μm. (N) Confocal imaging for Brca1 expression in NIH/3T3-EV and NIH/3T3-Ginir cells. Scale bars, 20 μm. (O) RNA-FISH using Ginir-specific probe (probe 1, FAM labelled) in NIH/3T3-EV cells visualised by confocal imaging. Scale bars, 20 μm. (P) Co-IP of Brca1 and Cep112 in NIH/3T3-Ginir(C) cells wherein lysates were treated independently with RNase (A, H, and III mix) or RNasin. Both RNase- and RNasin-treated lysates were immunoprecipitated with Brca1 antibody (sc-646, Santa Cruz) and blotted using Cep112 antibody (sc-246163, Santa Cruz). IP with anti-IgG served as control. Brca1, breast cancer type 1 susceptibility protein; Cep112, centrosomal protein 112; FAM, fluorescein amidite; Ginir, Genomic Instability Inducing RNA; IgG, immunoglobulin G; IP, immunoprecipitation; RIP, RNA-immunoprecipitation; RNasin, RNase inhibitor; RT-PCR, reverse transcription polymerase chain reaction; snRNA, small nuclear RNA.

Techniques Used: Western Blot, Binding Assay, Isolation, Reverse Transcription Polymerase Chain Reaction, In Silico, Generated, Co-Immunoprecipitation Assay, Over Expression, SDS Page, Staining, Imaging, Expressing, Fluorescence In Situ Hybridization, Immunoprecipitation

Expression of Ginir/Giniras transcripts during mouse embryonic development and in adult tissues. ). (B) RPA of RNA isolated from NIH/3T3 cells using PCR-generated sense or antisense riboprobes specific to Ginir sequence. Yeast total RNA served as control for RNase A/T1 activity. (C and D) Expression of Ginir/Giniras RNA in different stages of development (dpc) of mouse embryos (C) and in tissues from different organs of adult mice (D) using strand-specific cDNA synthesis and RT-PCR with G1F-G1R primers. Gapdh served as internal loading control. (E) Whole-mount ISH using LNA probes for Ginir (FAM labelled, green) or Giniras (TexRed labelled, red) on 10.5 dpc mouse embryos showing differential expression in brain (‘br’), forelimbs (‘fl’), and spinal cord (‘sc’). Whole-mount embryos treated with RNase A served as negative control for fluorescence. (F) FISH using LNA probes for Ginir (Green) or Giniras (Red) on embryo sections of 13.5 and 14.5 dpc embryos showing differential expression in forebrain (‘fb’), midbrain (‘mb’), hypothalamus (‘ht’), and limbs (‘li’). Embryo sections treated with RNase A served as a negative control. dpc, days post coitum; EST, expressed sequence tag; FAM, fluorescein amidite; FISH, fluorescence in situ hybridisation; Gapdh, glyceride 3-phosphate dehydrogenase; Ginir, Genomic Instability Inducing RNA; Giniras, antisense RNA of Ginir; ISH, in situ hybridisation; LNA, locked nucleic acid; PCR, polymerase chain reaction; RPA, ribonuclease protection assay; RT-PCR, reverse transcription PCR.
Figure Legend Snippet: Expression of Ginir/Giniras transcripts during mouse embryonic development and in adult tissues. ). (B) RPA of RNA isolated from NIH/3T3 cells using PCR-generated sense or antisense riboprobes specific to Ginir sequence. Yeast total RNA served as control for RNase A/T1 activity. (C and D) Expression of Ginir/Giniras RNA in different stages of development (dpc) of mouse embryos (C) and in tissues from different organs of adult mice (D) using strand-specific cDNA synthesis and RT-PCR with G1F-G1R primers. Gapdh served as internal loading control. (E) Whole-mount ISH using LNA probes for Ginir (FAM labelled, green) or Giniras (TexRed labelled, red) on 10.5 dpc mouse embryos showing differential expression in brain (‘br’), forelimbs (‘fl’), and spinal cord (‘sc’). Whole-mount embryos treated with RNase A served as negative control for fluorescence. (F) FISH using LNA probes for Ginir (Green) or Giniras (Red) on embryo sections of 13.5 and 14.5 dpc embryos showing differential expression in forebrain (‘fb’), midbrain (‘mb’), hypothalamus (‘ht’), and limbs (‘li’). Embryo sections treated with RNase A served as a negative control. dpc, days post coitum; EST, expressed sequence tag; FAM, fluorescein amidite; FISH, fluorescence in situ hybridisation; Gapdh, glyceride 3-phosphate dehydrogenase; Ginir, Genomic Instability Inducing RNA; Giniras, antisense RNA of Ginir; ISH, in situ hybridisation; LNA, locked nucleic acid; PCR, polymerase chain reaction; RPA, ribonuclease protection assay; RT-PCR, reverse transcription PCR.

Techniques Used: Expressing, Recombinase Polymerase Amplification, Isolation, Polymerase Chain Reaction, Generated, Sequencing, Activity Assay, Mouse Assay, Reverse Transcription Polymerase Chain Reaction, In Situ Hybridization, Negative Control, Fluorescence, Fluorescence In Situ Hybridization, In Situ, Hybridization

20) Product Images from "The systemic tumor response to RNase A treatment affects the expression of genes involved in maintaining cell malignancy"

Article Title: The systemic tumor response to RNase A treatment affects the expression of genes involved in maintaining cell malignancy

Journal: Oncotarget

doi: 10.18632/oncotarget.20228

Functional annotation of genes changed more than 1.4-fold in tumor tissue of LLC-bearing mice after treatment with RNase A based on gene ontology (GO) categorization (A) Biological Process [BP]. (B) Molecular Function [MF]. (C) Cellular Components [CC]. The green bars show the number of genes enriched in the sample of treated mice; red bars show the number of genes with decreased transcription in a group of treated mice.
Figure Legend Snippet: Functional annotation of genes changed more than 1.4-fold in tumor tissue of LLC-bearing mice after treatment with RNase A based on gene ontology (GO) categorization (A) Biological Process [BP]. (B) Molecular Function [MF]. (C) Cellular Components [CC]. The green bars show the number of genes enriched in the sample of treated mice; red bars show the number of genes with decreased transcription in a group of treated mice.

Techniques Used: Functional Assay, Mouse Assay

RT-qPCR analysis of expression levels of mRNA of Dusp6 , Fam89b , Map2k4 , Mtap , Serpinf1 , and Angptl4 genes in tumor tissue of LLC-bearing mice after treatment with RNase A The expression of mRNAs was normalized to Hprt1 and Ywhaz . Data are presented as mean ± SE. The level of the corresponding gene in the control (mice with LLC treated with saline buffer) was set at 1.
Figure Legend Snippet: RT-qPCR analysis of expression levels of mRNA of Dusp6 , Fam89b , Map2k4 , Mtap , Serpinf1 , and Angptl4 genes in tumor tissue of LLC-bearing mice after treatment with RNase A The expression of mRNAs was normalized to Hprt1 and Ywhaz . Data are presented as mean ± SE. The level of the corresponding gene in the control (mice with LLC treated with saline buffer) was set at 1.

Techniques Used: Quantitative RT-PCR, Expressing, Mouse Assay

Proposed mechanism of antitumor activity of RNase A RNase A therapy resulted in the boost of 116 miRNAs in tumour tissue and drop of 137 miRNAs in the bloodstream of mice with intramuscularly transplanted LLC and in the changes in the expression of 966 transcripts in tumor cells.
Figure Legend Snippet: Proposed mechanism of antitumor activity of RNase A RNase A therapy resulted in the boost of 116 miRNAs in tumour tissue and drop of 137 miRNAs in the bloodstream of mice with intramuscularly transplanted LLC and in the changes in the expression of 966 transcripts in tumor cells.

Techniques Used: Activity Assay, Mouse Assay, Expressing

Experimental design and data mining (A) Mice with i.m. transplanted LLC were treated with saline or RNase A at a dose of 0.7 μg/kg for 10 days starting on the 4 th day after tumor transplantation. At 1 h after the last injection, tumor tissue samples were collected. (B) Total RNA was isolated and pooled according to groups. mRNA fractions were enriched by ribosomal RNA depletion and used for the construction of cDNA libraries. Libraries were sequenced using the standard SOLiD™ V5.5 (Applied Biosystems) protocols. Reads were mapped to the Mus musculus reference genome (version NCBI37), and analysis of differential expression was performed. Differentially expressed transcripts were annotated to GO terms and analyzed using KEGG database to assign pathway mapping.
Figure Legend Snippet: Experimental design and data mining (A) Mice with i.m. transplanted LLC were treated with saline or RNase A at a dose of 0.7 μg/kg for 10 days starting on the 4 th day after tumor transplantation. At 1 h after the last injection, tumor tissue samples were collected. (B) Total RNA was isolated and pooled according to groups. mRNA fractions were enriched by ribosomal RNA depletion and used for the construction of cDNA libraries. Libraries were sequenced using the standard SOLiD™ V5.5 (Applied Biosystems) protocols. Reads were mapped to the Mus musculus reference genome (version NCBI37), and analysis of differential expression was performed. Differentially expressed transcripts were annotated to GO terms and analyzed using KEGG database to assign pathway mapping.

Techniques Used: Mouse Assay, Transplantation Assay, Injection, Isolation, Expressing

21) Product Images from "Noncoding RNA Ginir functions as an oncogene by associating with centrosomal proteins"

Article Title: Noncoding RNA Ginir functions as an oncogene by associating with centrosomal proteins

Journal: PLoS Biology

doi: 10.1371/journal.pbio.2004204

Ginir RNA impairs interaction between Cep112 and Brca1 proteins leading to genomic instability. (A) RNA pull-down with biotinylated Ginir RNA in NIH/3T3 cells followed by western blotting with Brca1 antibody (sc-646, Santa Cruz). Pull-down with unbiotinylated RNA probe served as control for nonspecific binding. (B-D) RIP performed using both Cep112 and Brca1 antibodies followed by RNA isolation and RT-PCR with Ginir specific primers (G2F-G2R) in NIH/3T3-GinirA (B) and NIH/3T3-GinirB (C) cells. RIP assay was also followed by RT-PCR using nonspecific primers for U6 snRNA (D). Anti-IgG IP served as control for nonspecific interaction. (E) In silico model of Cep112 and Brca1 interaction generated through computational docking using ZDOCK tool. (F and G) Co-IP of Cep112 and Brca1 proteins in NIH/3T3-EV cells. IP was performed with Brca1 antibody (sc-646, Santa Cruz) followed by immunoblotting with Cep112 antibody (sc-246162, Santa Cruz) (F) and vice versa (G). Anti-IgG IP served as a control for nonspecific binding to the antibody. (H and I) Western blotting with Cep112 antibody (24928-1-AP, Proteintech) for validation of Flag-Cep112 overexpression in NIH/3T3 cells (H). Fifty μg of whole-cell protein lysates from each of the mentioned cell lines was loaded on 7% SDS-PAGE. Tubulin served as internal loading control (I). (J) Co-IP of Brca1 and Flag-Cep112 in NIH-Flag-Cep112 cells. IP was performed with Flag1 antibody followed by immunoblotting with Brca1 antibody (20649-1-AP, Proteintech). Anti-IgG IP served as a control for nonspecific binding to the antibody. (K) Co-IP of Brca1 and Cep112 proteins in NIH/3T3-EV, NIH/3T3-Ginir, and NIH-Ginir-shGinir2 cells. IP was performed with Brca1 antibody (sc-646, Santa Cruz) followed by immunoblotting with Cep112 antibody (24928-1-AP, Proteintech). Anti-IgG IP served as a control for nonspecific binding to the antibody. (L) Confocal images showing colocalisation of Brca1 with γ-tubulin in NIH/3T3-EV cell line. Nuclei were stained with DAPI. Scale bars, 10 μm. (M) Confocal images showing colocalisation of Brca1 protein with Cep112 protein in NIH/3T3-EV cell line. Nuclei were stained with DAPI. Scale bars, 20 μm. (N) Confocal imaging for Brca1 expression in NIH/3T3-EV and NIH/3T3-Ginir cells. Scale bars, 20 μm. (O) RNA-FISH using Ginir-specific probe (probe 1, FAM labelled) in NIH/3T3-EV cells visualised by confocal imaging. Scale bars, 20 μm. (P) Co-IP of Brca1 and Cep112 in NIH/3T3-Ginir(C) cells wherein lysates were treated independently with RNase (A, H, and III mix) or RNasin. Both RNase- and RNasin-treated lysates were immunoprecipitated with Brca1 antibody (sc-646, Santa Cruz) and blotted using Cep112 antibody (sc-246163, Santa Cruz). IP with anti-IgG served as control. Brca1, breast cancer type 1 susceptibility protein; Cep112, centrosomal protein 112; FAM, fluorescein amidite; Ginir, Genomic Instability Inducing RNA; IgG, immunoglobulin G; IP, immunoprecipitation; RIP, RNA-immunoprecipitation; RNasin, RNase inhibitor; RT-PCR, reverse transcription polymerase chain reaction; snRNA, small nuclear RNA.
Figure Legend Snippet: Ginir RNA impairs interaction between Cep112 and Brca1 proteins leading to genomic instability. (A) RNA pull-down with biotinylated Ginir RNA in NIH/3T3 cells followed by western blotting with Brca1 antibody (sc-646, Santa Cruz). Pull-down with unbiotinylated RNA probe served as control for nonspecific binding. (B-D) RIP performed using both Cep112 and Brca1 antibodies followed by RNA isolation and RT-PCR with Ginir specific primers (G2F-G2R) in NIH/3T3-GinirA (B) and NIH/3T3-GinirB (C) cells. RIP assay was also followed by RT-PCR using nonspecific primers for U6 snRNA (D). Anti-IgG IP served as control for nonspecific interaction. (E) In silico model of Cep112 and Brca1 interaction generated through computational docking using ZDOCK tool. (F and G) Co-IP of Cep112 and Brca1 proteins in NIH/3T3-EV cells. IP was performed with Brca1 antibody (sc-646, Santa Cruz) followed by immunoblotting with Cep112 antibody (sc-246162, Santa Cruz) (F) and vice versa (G). Anti-IgG IP served as a control for nonspecific binding to the antibody. (H and I) Western blotting with Cep112 antibody (24928-1-AP, Proteintech) for validation of Flag-Cep112 overexpression in NIH/3T3 cells (H). Fifty μg of whole-cell protein lysates from each of the mentioned cell lines was loaded on 7% SDS-PAGE. Tubulin served as internal loading control (I). (J) Co-IP of Brca1 and Flag-Cep112 in NIH-Flag-Cep112 cells. IP was performed with Flag1 antibody followed by immunoblotting with Brca1 antibody (20649-1-AP, Proteintech). Anti-IgG IP served as a control for nonspecific binding to the antibody. (K) Co-IP of Brca1 and Cep112 proteins in NIH/3T3-EV, NIH/3T3-Ginir, and NIH-Ginir-shGinir2 cells. IP was performed with Brca1 antibody (sc-646, Santa Cruz) followed by immunoblotting with Cep112 antibody (24928-1-AP, Proteintech). Anti-IgG IP served as a control for nonspecific binding to the antibody. (L) Confocal images showing colocalisation of Brca1 with γ-tubulin in NIH/3T3-EV cell line. Nuclei were stained with DAPI. Scale bars, 10 μm. (M) Confocal images showing colocalisation of Brca1 protein with Cep112 protein in NIH/3T3-EV cell line. Nuclei were stained with DAPI. Scale bars, 20 μm. (N) Confocal imaging for Brca1 expression in NIH/3T3-EV and NIH/3T3-Ginir cells. Scale bars, 20 μm. (O) RNA-FISH using Ginir-specific probe (probe 1, FAM labelled) in NIH/3T3-EV cells visualised by confocal imaging. Scale bars, 20 μm. (P) Co-IP of Brca1 and Cep112 in NIH/3T3-Ginir(C) cells wherein lysates were treated independently with RNase (A, H, and III mix) or RNasin. Both RNase- and RNasin-treated lysates were immunoprecipitated with Brca1 antibody (sc-646, Santa Cruz) and blotted using Cep112 antibody (sc-246163, Santa Cruz). IP with anti-IgG served as control. Brca1, breast cancer type 1 susceptibility protein; Cep112, centrosomal protein 112; FAM, fluorescein amidite; Ginir, Genomic Instability Inducing RNA; IgG, immunoglobulin G; IP, immunoprecipitation; RIP, RNA-immunoprecipitation; RNasin, RNase inhibitor; RT-PCR, reverse transcription polymerase chain reaction; snRNA, small nuclear RNA.

Techniques Used: Western Blot, Binding Assay, Isolation, Reverse Transcription Polymerase Chain Reaction, In Silico, Generated, Co-Immunoprecipitation Assay, Over Expression, SDS Page, Staining, Imaging, Expressing, Fluorescence In Situ Hybridization, Immunoprecipitation

Expression of Ginir/Giniras transcripts during mouse embryonic development and in adult tissues. (A) List of several ESTs spanning Ginir sequence and showing significant similarity to Ginir ( http://blast.ncbi.nlm.nih.gov/Blast.cgi ). (B) RPA of RNA isolated from NIH/3T3 cells using PCR-generated sense or antisense riboprobes specific to Ginir sequence. Yeast total RNA served as control for RNase A/T1 activity. (C and D) Expression of Ginir/Giniras RNA in different stages of development (dpc) of mouse embryos (C) and in tissues from different organs of adult mice (D) using strand-specific cDNA synthesis and RT-PCR with G1F-G1R primers. Gapdh served as internal loading control. (E) Whole-mount ISH using LNA probes for Ginir (FAM labelled, green) or Giniras (TexRed labelled, red) on 10.5 dpc mouse embryos showing differential expression in brain (‘br’), forelimbs (‘fl’), and spinal cord (‘sc’). Whole-mount embryos treated with RNase A served as negative control for fluorescence. (F) FISH using LNA probes for Ginir (Green) or Giniras (Red) on embryo sections of 13.5 and 14.5 dpc embryos showing differential expression in forebrain (‘fb’), midbrain (‘mb’), hypothalamus (‘ht’), and limbs (‘li’). Embryo sections treated with RNase A served as a negative control. dpc, days post coitum; EST, expressed sequence tag; FAM, fluorescein amidite; FISH, fluorescence in situ hybridisation; Gapdh, glyceride 3-phosphate dehydrogenase; Ginir, Genomic Instability Inducing RNA; Giniras, antisense RNA of Ginir; ISH, in situ hybridisation; LNA, locked nucleic acid; PCR, polymerase chain reaction; RPA, ribonuclease protection assay; RT-PCR, reverse transcription PCR.
Figure Legend Snippet: Expression of Ginir/Giniras transcripts during mouse embryonic development and in adult tissues. (A) List of several ESTs spanning Ginir sequence and showing significant similarity to Ginir ( http://blast.ncbi.nlm.nih.gov/Blast.cgi ). (B) RPA of RNA isolated from NIH/3T3 cells using PCR-generated sense or antisense riboprobes specific to Ginir sequence. Yeast total RNA served as control for RNase A/T1 activity. (C and D) Expression of Ginir/Giniras RNA in different stages of development (dpc) of mouse embryos (C) and in tissues from different organs of adult mice (D) using strand-specific cDNA synthesis and RT-PCR with G1F-G1R primers. Gapdh served as internal loading control. (E) Whole-mount ISH using LNA probes for Ginir (FAM labelled, green) or Giniras (TexRed labelled, red) on 10.5 dpc mouse embryos showing differential expression in brain (‘br’), forelimbs (‘fl’), and spinal cord (‘sc’). Whole-mount embryos treated with RNase A served as negative control for fluorescence. (F) FISH using LNA probes for Ginir (Green) or Giniras (Red) on embryo sections of 13.5 and 14.5 dpc embryos showing differential expression in forebrain (‘fb’), midbrain (‘mb’), hypothalamus (‘ht’), and limbs (‘li’). Embryo sections treated with RNase A served as a negative control. dpc, days post coitum; EST, expressed sequence tag; FAM, fluorescein amidite; FISH, fluorescence in situ hybridisation; Gapdh, glyceride 3-phosphate dehydrogenase; Ginir, Genomic Instability Inducing RNA; Giniras, antisense RNA of Ginir; ISH, in situ hybridisation; LNA, locked nucleic acid; PCR, polymerase chain reaction; RPA, ribonuclease protection assay; RT-PCR, reverse transcription PCR.

Techniques Used: Expressing, Sequencing, Recombinase Polymerase Amplification, Isolation, Polymerase Chain Reaction, Generated, Activity Assay, Mouse Assay, Reverse Transcription Polymerase Chain Reaction, In Situ Hybridization, Negative Control, Fluorescence, Fluorescence In Situ Hybridization, In Situ, Hybridization

22) Product Images from "Noncoding RNA Ginir functions as an oncogene by associating with centrosomal proteins"

Article Title: Noncoding RNA Ginir functions as an oncogene by associating with centrosomal proteins

Journal: PLoS Biology

doi: 10.1371/journal.pbio.2004204

Ginir RNA impairs interaction between Cep112 and Brca1 proteins leading to genomic instability. (A) RNA pull-down with biotinylated Ginir RNA in NIH/3T3 cells followed by western blotting with Brca1 antibody (sc-646, Santa Cruz). Pull-down with unbiotinylated RNA probe served as control for nonspecific binding. (B-D) RIP performed using both Cep112 and Brca1 antibodies followed by RNA isolation and RT-PCR with Ginir specific primers (G2F-G2R) in NIH/3T3-GinirA (B) and NIH/3T3-GinirB (C) cells. RIP assay was also followed by RT-PCR using nonspecific primers for U6 snRNA (D). Anti-IgG IP served as control for nonspecific interaction. (E) In silico model of Cep112 and Brca1 interaction generated through computational docking using ZDOCK tool. (F and G) Co-IP of Cep112 and Brca1 proteins in NIH/3T3-EV cells. IP was performed with Brca1 antibody (sc-646, Santa Cruz) followed by immunoblotting with Cep112 antibody (sc-246162, Santa Cruz) (F) and vice versa (G). Anti-IgG IP served as a control for nonspecific binding to the antibody. (H and I) Western blotting with Cep112 antibody (24928-1-AP, Proteintech) for validation of Flag-Cep112 overexpression in NIH/3T3 cells (H). Fifty μg of whole-cell protein lysates from each of the mentioned cell lines was loaded on 7% SDS-PAGE. Tubulin served as internal loading control (I). (J) Co-IP of Brca1 and Flag-Cep112 in NIH-Flag-Cep112 cells. IP was performed with Flag1 antibody followed by immunoblotting with Brca1 antibody (20649-1-AP, Proteintech). Anti-IgG IP served as a control for nonspecific binding to the antibody. (K) Co-IP of Brca1 and Cep112 proteins in NIH/3T3-EV, NIH/3T3-Ginir, and NIH-Ginir-shGinir2 cells. IP was performed with Brca1 antibody (sc-646, Santa Cruz) followed by immunoblotting with Cep112 antibody (24928-1-AP, Proteintech). Anti-IgG IP served as a control for nonspecific binding to the antibody. (L) Confocal images showing colocalisation of Brca1 with γ-tubulin in NIH/3T3-EV cell line. Nuclei were stained with DAPI. Scale bars, 10 μm. (M) Confocal images showing colocalisation of Brca1 protein with Cep112 protein in NIH/3T3-EV cell line. Nuclei were stained with DAPI. Scale bars, 20 μm. (N) Confocal imaging for Brca1 expression in NIH/3T3-EV and NIH/3T3-Ginir cells. Scale bars, 20 μm. (O) RNA-FISH using Ginir-specific probe (probe 1, FAM labelled) in NIH/3T3-EV cells visualised by confocal imaging. Scale bars, 20 μm. (P) Co-IP of Brca1 and Cep112 in NIH/3T3-Ginir(C) cells wherein lysates were treated independently with RNase (A, H, and III mix) or RNasin. Both RNase- and RNasin-treated lysates were immunoprecipitated with Brca1 antibody (sc-646, Santa Cruz) and blotted using Cep112 antibody (sc-246163, Santa Cruz). IP with anti-IgG served as control. Brca1, breast cancer type 1 susceptibility protein; Cep112, centrosomal protein 112; FAM, fluorescein amidite; Ginir, Genomic Instability Inducing RNA; IgG, immunoglobulin G; IP, immunoprecipitation; RIP, RNA-immunoprecipitation; RNasin, RNase inhibitor; RT-PCR, reverse transcription polymerase chain reaction; snRNA, small nuclear RNA.
Figure Legend Snippet: Ginir RNA impairs interaction between Cep112 and Brca1 proteins leading to genomic instability. (A) RNA pull-down with biotinylated Ginir RNA in NIH/3T3 cells followed by western blotting with Brca1 antibody (sc-646, Santa Cruz). Pull-down with unbiotinylated RNA probe served as control for nonspecific binding. (B-D) RIP performed using both Cep112 and Brca1 antibodies followed by RNA isolation and RT-PCR with Ginir specific primers (G2F-G2R) in NIH/3T3-GinirA (B) and NIH/3T3-GinirB (C) cells. RIP assay was also followed by RT-PCR using nonspecific primers for U6 snRNA (D). Anti-IgG IP served as control for nonspecific interaction. (E) In silico model of Cep112 and Brca1 interaction generated through computational docking using ZDOCK tool. (F and G) Co-IP of Cep112 and Brca1 proteins in NIH/3T3-EV cells. IP was performed with Brca1 antibody (sc-646, Santa Cruz) followed by immunoblotting with Cep112 antibody (sc-246162, Santa Cruz) (F) and vice versa (G). Anti-IgG IP served as a control for nonspecific binding to the antibody. (H and I) Western blotting with Cep112 antibody (24928-1-AP, Proteintech) for validation of Flag-Cep112 overexpression in NIH/3T3 cells (H). Fifty μg of whole-cell protein lysates from each of the mentioned cell lines was loaded on 7% SDS-PAGE. Tubulin served as internal loading control (I). (J) Co-IP of Brca1 and Flag-Cep112 in NIH-Flag-Cep112 cells. IP was performed with Flag1 antibody followed by immunoblotting with Brca1 antibody (20649-1-AP, Proteintech). Anti-IgG IP served as a control for nonspecific binding to the antibody. (K) Co-IP of Brca1 and Cep112 proteins in NIH/3T3-EV, NIH/3T3-Ginir, and NIH-Ginir-shGinir2 cells. IP was performed with Brca1 antibody (sc-646, Santa Cruz) followed by immunoblotting with Cep112 antibody (24928-1-AP, Proteintech). Anti-IgG IP served as a control for nonspecific binding to the antibody. (L) Confocal images showing colocalisation of Brca1 with γ-tubulin in NIH/3T3-EV cell line. Nuclei were stained with DAPI. Scale bars, 10 μm. (M) Confocal images showing colocalisation of Brca1 protein with Cep112 protein in NIH/3T3-EV cell line. Nuclei were stained with DAPI. Scale bars, 20 μm. (N) Confocal imaging for Brca1 expression in NIH/3T3-EV and NIH/3T3-Ginir cells. Scale bars, 20 μm. (O) RNA-FISH using Ginir-specific probe (probe 1, FAM labelled) in NIH/3T3-EV cells visualised by confocal imaging. Scale bars, 20 μm. (P) Co-IP of Brca1 and Cep112 in NIH/3T3-Ginir(C) cells wherein lysates were treated independently with RNase (A, H, and III mix) or RNasin. Both RNase- and RNasin-treated lysates were immunoprecipitated with Brca1 antibody (sc-646, Santa Cruz) and blotted using Cep112 antibody (sc-246163, Santa Cruz). IP with anti-IgG served as control. Brca1, breast cancer type 1 susceptibility protein; Cep112, centrosomal protein 112; FAM, fluorescein amidite; Ginir, Genomic Instability Inducing RNA; IgG, immunoglobulin G; IP, immunoprecipitation; RIP, RNA-immunoprecipitation; RNasin, RNase inhibitor; RT-PCR, reverse transcription polymerase chain reaction; snRNA, small nuclear RNA.

Techniques Used: Western Blot, Binding Assay, Isolation, Reverse Transcription Polymerase Chain Reaction, In Silico, Generated, Co-Immunoprecipitation Assay, Over Expression, SDS Page, Staining, Imaging, Expressing, Fluorescence In Situ Hybridization, Immunoprecipitation

Expression of Ginir/Giniras transcripts during mouse embryonic development and in adult tissues. (A) List of several ESTs spanning Ginir sequence and showing significant similarity to Ginir ( http://blast.ncbi.nlm.nih.gov/Blast.cgi ). (B) RPA of RNA isolated from NIH/3T3 cells using PCR-generated sense or antisense riboprobes specific to Ginir sequence. Yeast total RNA served as control for RNase A/T1 activity. (C and D) Expression of Ginir/Giniras RNA in different stages of development (dpc) of mouse embryos (C) and in tissues from different organs of adult mice (D) using strand-specific cDNA synthesis and RT-PCR with G1F-G1R primers. Gapdh served as internal loading control. (E) Whole-mount ISH using LNA probes for Ginir (FAM labelled, green) or Giniras (TexRed labelled, red) on 10.5 dpc mouse embryos showing differential expression in brain (‘br’), forelimbs (‘fl’), and spinal cord (‘sc’). Whole-mount embryos treated with RNase A served as negative control for fluorescence. (F) FISH using LNA probes for Ginir (Green) or Giniras (Red) on embryo sections of 13.5 and 14.5 dpc embryos showing differential expression in forebrain (‘fb’), midbrain (‘mb’), hypothalamus (‘ht’), and limbs (‘li’). Embryo sections treated with RNase A served as a negative control. dpc, days post coitum; EST, expressed sequence tag; FAM, fluorescein amidite; FISH, fluorescence in situ hybridisation; Gapdh, glyceride 3-phosphate dehydrogenase; Ginir, Genomic Instability Inducing RNA; Giniras, antisense RNA of Ginir; ISH, in situ hybridisation; LNA, locked nucleic acid; PCR, polymerase chain reaction; RPA, ribonuclease protection assay; RT-PCR, reverse transcription PCR.
Figure Legend Snippet: Expression of Ginir/Giniras transcripts during mouse embryonic development and in adult tissues. (A) List of several ESTs spanning Ginir sequence and showing significant similarity to Ginir ( http://blast.ncbi.nlm.nih.gov/Blast.cgi ). (B) RPA of RNA isolated from NIH/3T3 cells using PCR-generated sense or antisense riboprobes specific to Ginir sequence. Yeast total RNA served as control for RNase A/T1 activity. (C and D) Expression of Ginir/Giniras RNA in different stages of development (dpc) of mouse embryos (C) and in tissues from different organs of adult mice (D) using strand-specific cDNA synthesis and RT-PCR with G1F-G1R primers. Gapdh served as internal loading control. (E) Whole-mount ISH using LNA probes for Ginir (FAM labelled, green) or Giniras (TexRed labelled, red) on 10.5 dpc mouse embryos showing differential expression in brain (‘br’), forelimbs (‘fl’), and spinal cord (‘sc’). Whole-mount embryos treated with RNase A served as negative control for fluorescence. (F) FISH using LNA probes for Ginir (Green) or Giniras (Red) on embryo sections of 13.5 and 14.5 dpc embryos showing differential expression in forebrain (‘fb’), midbrain (‘mb’), hypothalamus (‘ht’), and limbs (‘li’). Embryo sections treated with RNase A served as a negative control. dpc, days post coitum; EST, expressed sequence tag; FAM, fluorescein amidite; FISH, fluorescence in situ hybridisation; Gapdh, glyceride 3-phosphate dehydrogenase; Ginir, Genomic Instability Inducing RNA; Giniras, antisense RNA of Ginir; ISH, in situ hybridisation; LNA, locked nucleic acid; PCR, polymerase chain reaction; RPA, ribonuclease protection assay; RT-PCR, reverse transcription PCR.

Techniques Used: Expressing, Sequencing, Recombinase Polymerase Amplification, Isolation, Polymerase Chain Reaction, Generated, Activity Assay, Mouse Assay, Reverse Transcription Polymerase Chain Reaction, In Situ Hybridization, Negative Control, Fluorescence, Fluorescence In Situ Hybridization, In Situ, Hybridization

23) Product Images from "Noncoding RNA Ginir functions as an oncogene by associating with centrosomal proteins"

Article Title: Noncoding RNA Ginir functions as an oncogene by associating with centrosomal proteins

Journal: PLoS Biology

doi: 10.1371/journal.pbio.2004204

Ginir RNA impairs interaction between Cep112 and Brca1 proteins leading to genomic instability. (A) RNA pull-down with biotinylated Ginir RNA in NIH/3T3 cells followed by western blotting with Brca1 antibody (sc-646, Santa Cruz). Pull-down with unbiotinylated RNA probe served as control for nonspecific binding. (B-D) RIP performed using both Cep112 and Brca1 antibodies followed by RNA isolation and RT-PCR with Ginir specific primers (G2F-G2R) in NIH/3T3-GinirA (B) and NIH/3T3-GinirB (C) cells. RIP assay was also followed by RT-PCR using nonspecific primers for U6 snRNA (D). Anti-IgG IP served as control for nonspecific interaction. (E) In silico model of Cep112 and Brca1 interaction generated through computational docking using ZDOCK tool. (F and G) Co-IP of Cep112 and Brca1 proteins in NIH/3T3-EV cells. IP was performed with Brca1 antibody (sc-646, Santa Cruz) followed by immunoblotting with Cep112 antibody (sc-246162, Santa Cruz) (F) and vice versa (G). Anti-IgG IP served as a control for nonspecific binding to the antibody. (H and I) Western blotting with Cep112 antibody (24928-1-AP, Proteintech) for validation of Flag-Cep112 overexpression in NIH/3T3 cells (H). Fifty μg of whole-cell protein lysates from each of the mentioned cell lines was loaded on 7% SDS-PAGE. Tubulin served as internal loading control (I). (J) Co-IP of Brca1 and Flag-Cep112 in NIH-Flag-Cep112 cells. IP was performed with Flag1 antibody followed by immunoblotting with Brca1 antibody (20649-1-AP, Proteintech). Anti-IgG IP served as a control for nonspecific binding to the antibody. (K) Co-IP of Brca1 and Cep112 proteins in NIH/3T3-EV, NIH/3T3-Ginir, and NIH-Ginir-shGinir2 cells. IP was performed with Brca1 antibody (sc-646, Santa Cruz) followed by immunoblotting with Cep112 antibody (24928-1-AP, Proteintech). Anti-IgG IP served as a control for nonspecific binding to the antibody. (L) Confocal images showing colocalisation of Brca1 with γ-tubulin in NIH/3T3-EV cell line. Nuclei were stained with DAPI. Scale bars, 10 μm. (M) Confocal images showing colocalisation of Brca1 protein with Cep112 protein in NIH/3T3-EV cell line. Nuclei were stained with DAPI. Scale bars, 20 μm. (N) Confocal imaging for Brca1 expression in NIH/3T3-EV and NIH/3T3-Ginir cells. Scale bars, 20 μm. (O) RNA-FISH using Ginir-specific probe (probe 1, FAM labelled) in NIH/3T3-EV cells visualised by confocal imaging. Scale bars, 20 μm. (P) Co-IP of Brca1 and Cep112 in NIH/3T3-Ginir(C) cells wherein lysates were treated independently with RNase (A, H, and III mix) or RNasin. Both RNase- and RNasin-treated lysates were immunoprecipitated with Brca1 antibody (sc-646, Santa Cruz) and blotted using Cep112 antibody (sc-246163, Santa Cruz). IP with anti-IgG served as control. Brca1, breast cancer type 1 susceptibility protein; Cep112, centrosomal protein 112; FAM, fluorescein amidite; Ginir, Genomic Instability Inducing RNA; IgG, immunoglobulin G; IP, immunoprecipitation; RIP, RNA-immunoprecipitation; RNasin, RNase inhibitor; RT-PCR, reverse transcription polymerase chain reaction; snRNA, small nuclear RNA.
Figure Legend Snippet: Ginir RNA impairs interaction between Cep112 and Brca1 proteins leading to genomic instability. (A) RNA pull-down with biotinylated Ginir RNA in NIH/3T3 cells followed by western blotting with Brca1 antibody (sc-646, Santa Cruz). Pull-down with unbiotinylated RNA probe served as control for nonspecific binding. (B-D) RIP performed using both Cep112 and Brca1 antibodies followed by RNA isolation and RT-PCR with Ginir specific primers (G2F-G2R) in NIH/3T3-GinirA (B) and NIH/3T3-GinirB (C) cells. RIP assay was also followed by RT-PCR using nonspecific primers for U6 snRNA (D). Anti-IgG IP served as control for nonspecific interaction. (E) In silico model of Cep112 and Brca1 interaction generated through computational docking using ZDOCK tool. (F and G) Co-IP of Cep112 and Brca1 proteins in NIH/3T3-EV cells. IP was performed with Brca1 antibody (sc-646, Santa Cruz) followed by immunoblotting with Cep112 antibody (sc-246162, Santa Cruz) (F) and vice versa (G). Anti-IgG IP served as a control for nonspecific binding to the antibody. (H and I) Western blotting with Cep112 antibody (24928-1-AP, Proteintech) for validation of Flag-Cep112 overexpression in NIH/3T3 cells (H). Fifty μg of whole-cell protein lysates from each of the mentioned cell lines was loaded on 7% SDS-PAGE. Tubulin served as internal loading control (I). (J) Co-IP of Brca1 and Flag-Cep112 in NIH-Flag-Cep112 cells. IP was performed with Flag1 antibody followed by immunoblotting with Brca1 antibody (20649-1-AP, Proteintech). Anti-IgG IP served as a control for nonspecific binding to the antibody. (K) Co-IP of Brca1 and Cep112 proteins in NIH/3T3-EV, NIH/3T3-Ginir, and NIH-Ginir-shGinir2 cells. IP was performed with Brca1 antibody (sc-646, Santa Cruz) followed by immunoblotting with Cep112 antibody (24928-1-AP, Proteintech). Anti-IgG IP served as a control for nonspecific binding to the antibody. (L) Confocal images showing colocalisation of Brca1 with γ-tubulin in NIH/3T3-EV cell line. Nuclei were stained with DAPI. Scale bars, 10 μm. (M) Confocal images showing colocalisation of Brca1 protein with Cep112 protein in NIH/3T3-EV cell line. Nuclei were stained with DAPI. Scale bars, 20 μm. (N) Confocal imaging for Brca1 expression in NIH/3T3-EV and NIH/3T3-Ginir cells. Scale bars, 20 μm. (O) RNA-FISH using Ginir-specific probe (probe 1, FAM labelled) in NIH/3T3-EV cells visualised by confocal imaging. Scale bars, 20 μm. (P) Co-IP of Brca1 and Cep112 in NIH/3T3-Ginir(C) cells wherein lysates were treated independently with RNase (A, H, and III mix) or RNasin. Both RNase- and RNasin-treated lysates were immunoprecipitated with Brca1 antibody (sc-646, Santa Cruz) and blotted using Cep112 antibody (sc-246163, Santa Cruz). IP with anti-IgG served as control. Brca1, breast cancer type 1 susceptibility protein; Cep112, centrosomal protein 112; FAM, fluorescein amidite; Ginir, Genomic Instability Inducing RNA; IgG, immunoglobulin G; IP, immunoprecipitation; RIP, RNA-immunoprecipitation; RNasin, RNase inhibitor; RT-PCR, reverse transcription polymerase chain reaction; snRNA, small nuclear RNA.

Techniques Used: Western Blot, Binding Assay, Isolation, Reverse Transcription Polymerase Chain Reaction, In Silico, Generated, Co-Immunoprecipitation Assay, Over Expression, SDS Page, Staining, Imaging, Expressing, Fluorescence In Situ Hybridization, Immunoprecipitation

Expression of Ginir/Giniras transcripts during mouse embryonic development and in adult tissues. ). (B) RPA of RNA isolated from NIH/3T3 cells using PCR-generated sense or antisense riboprobes specific to Ginir sequence. Yeast total RNA served as control for RNase A/T1 activity. (C and D) Expression of Ginir/Giniras RNA in different stages of development (dpc) of mouse embryos (C) and in tissues from different organs of adult mice (D) using strand-specific cDNA synthesis and RT-PCR with G1F-G1R primers. Gapdh served as internal loading control. (E) Whole-mount ISH using LNA probes for Ginir (FAM labelled, green) or Giniras (TexRed labelled, red) on 10.5 dpc mouse embryos showing differential expression in brain (‘br’), forelimbs (‘fl’), and spinal cord (‘sc’). Whole-mount embryos treated with RNase A served as negative control for fluorescence. (F) FISH using LNA probes for Ginir (Green) or Giniras (Red) on embryo sections of 13.5 and 14.5 dpc embryos showing differential expression in forebrain (‘fb’), midbrain (‘mb’), hypothalamus (‘ht’), and limbs (‘li’). Embryo sections treated with RNase A served as a negative control. dpc, days post coitum; EST, expressed sequence tag; FAM, fluorescein amidite; FISH, fluorescence in situ hybridisation; Gapdh, glyceride 3-phosphate dehydrogenase; Ginir, Genomic Instability Inducing RNA; Giniras, antisense RNA of Ginir; ISH, in situ hybridisation; LNA, locked nucleic acid; PCR, polymerase chain reaction; RPA, ribonuclease protection assay; RT-PCR, reverse transcription PCR.
Figure Legend Snippet: Expression of Ginir/Giniras transcripts during mouse embryonic development and in adult tissues. ). (B) RPA of RNA isolated from NIH/3T3 cells using PCR-generated sense or antisense riboprobes specific to Ginir sequence. Yeast total RNA served as control for RNase A/T1 activity. (C and D) Expression of Ginir/Giniras RNA in different stages of development (dpc) of mouse embryos (C) and in tissues from different organs of adult mice (D) using strand-specific cDNA synthesis and RT-PCR with G1F-G1R primers. Gapdh served as internal loading control. (E) Whole-mount ISH using LNA probes for Ginir (FAM labelled, green) or Giniras (TexRed labelled, red) on 10.5 dpc mouse embryos showing differential expression in brain (‘br’), forelimbs (‘fl’), and spinal cord (‘sc’). Whole-mount embryos treated with RNase A served as negative control for fluorescence. (F) FISH using LNA probes for Ginir (Green) or Giniras (Red) on embryo sections of 13.5 and 14.5 dpc embryos showing differential expression in forebrain (‘fb’), midbrain (‘mb’), hypothalamus (‘ht’), and limbs (‘li’). Embryo sections treated with RNase A served as a negative control. dpc, days post coitum; EST, expressed sequence tag; FAM, fluorescein amidite; FISH, fluorescence in situ hybridisation; Gapdh, glyceride 3-phosphate dehydrogenase; Ginir, Genomic Instability Inducing RNA; Giniras, antisense RNA of Ginir; ISH, in situ hybridisation; LNA, locked nucleic acid; PCR, polymerase chain reaction; RPA, ribonuclease protection assay; RT-PCR, reverse transcription PCR.

Techniques Used: Expressing, Recombinase Polymerase Amplification, Isolation, Polymerase Chain Reaction, Generated, Sequencing, Activity Assay, Mouse Assay, Reverse Transcription Polymerase Chain Reaction, In Situ Hybridization, Negative Control, Fluorescence, Fluorescence In Situ Hybridization, In Situ, Hybridization

24) Product Images from "Unexpected DNA Loss Mediated by the DNA Binding Activity of Ribonuclease A"

Article Title: Unexpected DNA Loss Mediated by the DNA Binding Activity of Ribonuclease A

Journal: PLoS ONE

doi: 10.1371/journal.pone.0115008

RNase and DNase analysis of major satellite species. Total RNA from NIH/3T3 cells was treated with indicated enzymes for 30 min at 37°, phenol:chloroform extracted, ethanol precipitated and glyoxylated prior to electrophoresis, then blotted and probed for major satellite sequences, ethidium staining of ribosomal RNA is shown as a control. A: Samples treated in water without and with RNase A. B: Samples treated in NEBuffer 3 with indicated ribonucleases. C: Samples treated in RQ1 DNase buffer without and with DNase I. For quantification, error bars represent ±1 s.d., y-axes in arbitrary units, p values calculated using a one-way ANOVA (n = 4) for B and Student’s  t -test (n = 3) for C.
Figure Legend Snippet: RNase and DNase analysis of major satellite species. Total RNA from NIH/3T3 cells was treated with indicated enzymes for 30 min at 37°, phenol:chloroform extracted, ethanol precipitated and glyoxylated prior to electrophoresis, then blotted and probed for major satellite sequences, ethidium staining of ribosomal RNA is shown as a control. A: Samples treated in water without and with RNase A. B: Samples treated in NEBuffer 3 with indicated ribonucleases. C: Samples treated in RQ1 DNase buffer without and with DNase I. For quantification, error bars represent ±1 s.d., y-axes in arbitrary units, p values calculated using a one-way ANOVA (n = 4) for B and Student’s t -test (n = 3) for C.

Techniques Used: Electrophoresis, Staining

Inhibition of DNA removal by RNase A. A: 0.5 ng major satellite PCR product was mixed with 1 µg NIH/3T3 RNA (lanes 1, 2) or with 1 µg DNA molecular weight marker (lanes 3, 4), and treated without or with RNase A followed by phenol:chloroform extraction and analysis as in Fig. 1. B: Identical to A, except that samples were incubated with 20 µg proteinase K for 15 min at 37° after RNase A treatment and phenol:chloroform extraction was omitted. For quantification, error bars represent ±1 s.d., y-axes in arbitrary units, analysis by one-way ANOVA (n = 3), differences in B are not significant.
Figure Legend Snippet: Inhibition of DNA removal by RNase A. A: 0.5 ng major satellite PCR product was mixed with 1 µg NIH/3T3 RNA (lanes 1, 2) or with 1 µg DNA molecular weight marker (lanes 3, 4), and treated without or with RNase A followed by phenol:chloroform extraction and analysis as in Fig. 1. B: Identical to A, except that samples were incubated with 20 µg proteinase K for 15 min at 37° after RNase A treatment and phenol:chloroform extraction was omitted. For quantification, error bars represent ±1 s.d., y-axes in arbitrary units, analysis by one-way ANOVA (n = 3), differences in B are not significant.

Techniques Used: Inhibition, Polymerase Chain Reaction, Molecular Weight, Marker, Incubation

RNase A activity on DNA. A, B: 0.5 ng major satellite PCR product was mixed with 1 µg NIH/3T3 total RNA and analysed as in Fig. 1. A: Treatment with RNase A from different manufacturers. B: Treatment with RNase A in different buffers. C: 50 ng DNA molecular weight marker was mixed with 1 µg NIH/3T3 total RNA and treated without or with RNase A, purified as in Fig. 1 and separated on a non-denaturing 1xTBE gel before blotting and probing for the molecular weight marker. D: 32 P-labelled 50 bp ladder mixed with 1 µg RNA was treated without or with RNase A for 30 min at 37°, directly separated on an 8% PAGE gel and exposed to a phosphorimaging screen. For quantification, error bars represent ±1 s.d., y-axes in arbitrary units, p values were calculated by one-way ANOVA (A, B) or Student’s t -test (C, D), n = 3 in all cases.
Figure Legend Snippet: RNase A activity on DNA. A, B: 0.5 ng major satellite PCR product was mixed with 1 µg NIH/3T3 total RNA and analysed as in Fig. 1. A: Treatment with RNase A from different manufacturers. B: Treatment with RNase A in different buffers. C: 50 ng DNA molecular weight marker was mixed with 1 µg NIH/3T3 total RNA and treated without or with RNase A, purified as in Fig. 1 and separated on a non-denaturing 1xTBE gel before blotting and probing for the molecular weight marker. D: 32 P-labelled 50 bp ladder mixed with 1 µg RNA was treated without or with RNase A for 30 min at 37°, directly separated on an 8% PAGE gel and exposed to a phosphorimaging screen. For quantification, error bars represent ±1 s.d., y-axes in arbitrary units, p values were calculated by one-way ANOVA (A, B) or Student’s t -test (C, D), n = 3 in all cases.

Techniques Used: Activity Assay, Polymerase Chain Reaction, Molecular Weight, Marker, Purification, Polyacrylamide Gel Electrophoresis

Re-partitioning of RNase-treated DNA. A, B: 5 ng 32 P-labelled major satellite PCR product mixed with 1 µg of NIH/3T3 RNA was incubated without or with RNase A for 30 min at 37°. Reactions were then diluted to 100 µl with water and extracted with 100 µl phenol:chloroform. A: 10 µl of the aqueous phase was analysed by electrophoresis in a non-denaturing 1xTBE gel which was dried and exposed to a phosphorimaging screen. B: Radioactivity present in the aqueous and phenol phases quantitated using a Geiger counter. C: DNA loss after RNase A treatment with phenol:chloroform extraction at pH 7 or pH 8. D: Activity of RNase A in phenol:chloroform. 1 µl 1 mg/ml RNase A was added to 100 µl phenol:chloroform and vortexed. 1 µl 1 mg/ml NIH/3T3 RNA was added, vortexed and reactions incubated at 37° for 30 min, followed by extraction with 100 µl water, precipitation and gel electrophoresis. For quantification, error bars represent ±1 s.d, y-axes in arbitrary units for A, D or Bequerels in B, analysis by Student’s t -test, n = 3.
Figure Legend Snippet: Re-partitioning of RNase-treated DNA. A, B: 5 ng 32 P-labelled major satellite PCR product mixed with 1 µg of NIH/3T3 RNA was incubated without or with RNase A for 30 min at 37°. Reactions were then diluted to 100 µl with water and extracted with 100 µl phenol:chloroform. A: 10 µl of the aqueous phase was analysed by electrophoresis in a non-denaturing 1xTBE gel which was dried and exposed to a phosphorimaging screen. B: Radioactivity present in the aqueous and phenol phases quantitated using a Geiger counter. C: DNA loss after RNase A treatment with phenol:chloroform extraction at pH 7 or pH 8. D: Activity of RNase A in phenol:chloroform. 1 µl 1 mg/ml RNase A was added to 100 µl phenol:chloroform and vortexed. 1 µl 1 mg/ml NIH/3T3 RNA was added, vortexed and reactions incubated at 37° for 30 min, followed by extraction with 100 µl water, precipitation and gel electrophoresis. For quantification, error bars represent ±1 s.d, y-axes in arbitrary units for A, D or Bequerels in B, analysis by Student’s t -test, n = 3.

Techniques Used: Polymerase Chain Reaction, Incubation, Electrophoresis, Radioactivity, Activity Assay, Nucleic Acid Electrophoresis

25) Product Images from "Mammalian splicing factor SF1 interacts with SURP domains of U2 snRNP-associated proteins"

Article Title: Mammalian splicing factor SF1 interacts with SURP domains of U2 snRNP-associated proteins

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkv952

Determination of the SURP-ID of SF1. ( A ) Scheme of mutant SF1 proteins. Boxes represent known protein domains: ULM, UHM ligand motif; KH/QUA2, K-homology/Quaking2 domain; Zn, zinc knuckle. Numbering above full-length SF1 refers to amino acids. The names of SF1 mutants are shown on the left; numbers on the right refer to the residues comprising the proteins. ( B ) and ( C ) GST pull-down of mutant SF1 proteins with SURP domains. GST-tagged SF3a120-SURP1, CHERP-SURP, SFSWAP-SURP2, U2AF65-UHM and GST alone, bound to glutathione agarose (as indicated on the right), were incubated with mutant His 6 - (B) or His 6 -MBP-tagged (C) SF1 proteins as indicated above the figures. Bound proteins were separated by 10% SDS-PAGE and western blotted with anti-His 6  (top panels) and anti-GST (bottom panels). The input (10% of the total) is shown at the bottom. ( D ) Co-IP. GFP-tagged SF1-C370 and -C302 were transiently expressed in HeLa cells. Total cell lysates were RNase A-treated and incubated with Dynabeads Protein G-coupled anti-GFP. Input (I; 0.5% of the total) and bound proteins (B) were separated by 7.5% SDS-PAGE and western blotted with anti-SF3a120, anti-U2AF65 and anti-GFP as indicated on the right. The migration of protein markers (in kDa) is shown on the left. ( E ) Multiple sequence alignment of SF1 proteins. SF1 sequences taken from the UniProt database ( www.uniprot.org ) were aligned with ClustalW2 ( www.ebi.ac.uk/Tools/msa/clustalw2/ ). The region from the zinc knuckle of human SF1 and 40 amino acids C-terminal of this domain is shown. Numbering is given for human SF1. Amino acids identical in more than 50% of the sequences are marked.
Figure Legend Snippet: Determination of the SURP-ID of SF1. ( A ) Scheme of mutant SF1 proteins. Boxes represent known protein domains: ULM, UHM ligand motif; KH/QUA2, K-homology/Quaking2 domain; Zn, zinc knuckle. Numbering above full-length SF1 refers to amino acids. The names of SF1 mutants are shown on the left; numbers on the right refer to the residues comprising the proteins. ( B ) and ( C ) GST pull-down of mutant SF1 proteins with SURP domains. GST-tagged SF3a120-SURP1, CHERP-SURP, SFSWAP-SURP2, U2AF65-UHM and GST alone, bound to glutathione agarose (as indicated on the right), were incubated with mutant His 6 - (B) or His 6 -MBP-tagged (C) SF1 proteins as indicated above the figures. Bound proteins were separated by 10% SDS-PAGE and western blotted with anti-His 6 (top panels) and anti-GST (bottom panels). The input (10% of the total) is shown at the bottom. ( D ) Co-IP. GFP-tagged SF1-C370 and -C302 were transiently expressed in HeLa cells. Total cell lysates were RNase A-treated and incubated with Dynabeads Protein G-coupled anti-GFP. Input (I; 0.5% of the total) and bound proteins (B) were separated by 7.5% SDS-PAGE and western blotted with anti-SF3a120, anti-U2AF65 and anti-GFP as indicated on the right. The migration of protein markers (in kDa) is shown on the left. ( E ) Multiple sequence alignment of SF1 proteins. SF1 sequences taken from the UniProt database ( www.uniprot.org ) were aligned with ClustalW2 ( www.ebi.ac.uk/Tools/msa/clustalw2/ ). The region from the zinc knuckle of human SF1 and 40 amino acids C-terminal of this domain is shown. Numbering is given for human SF1. Amino acids identical in more than 50% of the sequences are marked.

Techniques Used: Mutagenesis, Incubation, SDS Page, Western Blot, Co-Immunoprecipitation Assay, Migration, Sequencing

Deletion of the SF1 SURP-ID does not reduce U2AF65 binding to the pre-mRNA. ( A ) and ( B ) U2AF65 UV cross-linking to AdML 3′ splice site substrates. Splicing reactions containing radio-labeled RNA with a consensus (A) or weak (B) BPS, mock or SF1-depleted (ΔSF1) extracts complemented with 2.2 pmole His 6 -SF1-C370 or -C302 as indicated were incubated at 30°C for 15 min. Samples were UV cross-linked, RNase A-treated and immunoprecipitated with control IgG or anti-U2AF65, as indicated. RNA–protein complexes were separated by 10% SDS-PAGE. Gels were dried and exposed to PhosphorImager screens. The top panels show representative results of triplicate experiments; quantifications are shown in the bottom panels. ‘% U2AF65-bound RNA’ indicates the percentage of the intensity of the cross-linked RNAs normalized to the RNA immunoprecipitated with anti-U2AF65 from mock-treated extract. Data are shown as mean value ± SEM.
Figure Legend Snippet: Deletion of the SF1 SURP-ID does not reduce U2AF65 binding to the pre-mRNA. ( A ) and ( B ) U2AF65 UV cross-linking to AdML 3′ splice site substrates. Splicing reactions containing radio-labeled RNA with a consensus (A) or weak (B) BPS, mock or SF1-depleted (ΔSF1) extracts complemented with 2.2 pmole His 6 -SF1-C370 or -C302 as indicated were incubated at 30°C for 15 min. Samples were UV cross-linked, RNase A-treated and immunoprecipitated with control IgG or anti-U2AF65, as indicated. RNA–protein complexes were separated by 10% SDS-PAGE. Gels were dried and exposed to PhosphorImager screens. The top panels show representative results of triplicate experiments; quantifications are shown in the bottom panels. ‘% U2AF65-bound RNA’ indicates the percentage of the intensity of the cross-linked RNAs normalized to the RNA immunoprecipitated with anti-U2AF65 from mock-treated extract. Data are shown as mean value ± SEM.

Techniques Used: Binding Assay, Labeling, Incubation, Immunoprecipitation, SDS Page

Analysis of the interaction of SF1 with SURP domain-containing proteins. ( A ) Scheme of SURP domain-containing proteins identified in Y2H screens. The domain structure of SF3a120, CHERP and SFSWAP is shown with SURP domains indicated in dark grey. Other domains are shown in light grey: CID, RNA polymerase II-binding domain; G-patch, G-patch domain; RS domain, Arg/Ser-rich domain; UBL, ubiquitin-like domain. Numbering was taken from UniProt entries ( www.uniprot.org ). The smallest selected interaction domain (ΣSID) deduced from cDNAs found in the Y2H screens is indicated below the proteins (numbering according to human prote ins). ( B ) Co-IP. HeLa cell nuclear extract was incubated with Dynabeads Protein G coated with anti-SF1 or control IgG. Input (I; 10% of total), bound (B) and unbound (U) fractions were separated by 7.5% SDS-PAGE (10% for anti-H1) followed by western blotting with antibodies against the proteins indicated on the right side of each panel. ( C ) GST pull-down. His 6 -tagged SF1-C370 was incubated with GST alone, GST-tagged U2AF65-UHM, SF3a120-SURP1, CHERP-SURP or SFSWAP-SURP2 bound to glutathione-agarose as indicated above the figure. GST-tagged proteins were mock-treated (−) or digested with RNase A (+) as shown. The His 6 -SF1-C370 input (I; 10% of total) and bound proteins were separated by 10% SDS-PAGE and western blotted with anti-His 6 (top) and anti-GST antibodies (bottom). The migration of protein markers is indicated in kDa on the left.
Figure Legend Snippet: Analysis of the interaction of SF1 with SURP domain-containing proteins. ( A ) Scheme of SURP domain-containing proteins identified in Y2H screens. The domain structure of SF3a120, CHERP and SFSWAP is shown with SURP domains indicated in dark grey. Other domains are shown in light grey: CID, RNA polymerase II-binding domain; G-patch, G-patch domain; RS domain, Arg/Ser-rich domain; UBL, ubiquitin-like domain. Numbering was taken from UniProt entries ( www.uniprot.org ). The smallest selected interaction domain (ΣSID) deduced from cDNAs found in the Y2H screens is indicated below the proteins (numbering according to human prote ins). ( B ) Co-IP. HeLa cell nuclear extract was incubated with Dynabeads Protein G coated with anti-SF1 or control IgG. Input (I; 10% of total), bound (B) and unbound (U) fractions were separated by 7.5% SDS-PAGE (10% for anti-H1) followed by western blotting with antibodies against the proteins indicated on the right side of each panel. ( C ) GST pull-down. His 6 -tagged SF1-C370 was incubated with GST alone, GST-tagged U2AF65-UHM, SF3a120-SURP1, CHERP-SURP or SFSWAP-SURP2 bound to glutathione-agarose as indicated above the figure. GST-tagged proteins were mock-treated (−) or digested with RNase A (+) as shown. The His 6 -SF1-C370 input (I; 10% of total) and bound proteins were separated by 10% SDS-PAGE and western blotted with anti-His 6 (top) and anti-GST antibodies (bottom). The migration of protein markers is indicated in kDa on the left.

Techniques Used: Binding Assay, Co-Immunoprecipitation Assay, Incubation, SDS Page, Western Blot, Migration

26) Product Images from "Transcription of telomeric DNA leads to high levels of homologous recombination and t-loops"

Article Title: Transcription of telomeric DNA leads to high levels of homologous recombination and t-loops

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkw779

Agarose gel electrophoretic analysis of DNA transcribed in the presence of TRF1 or TRF2. ( A ) Agarose gel electrophoresis of linear pRST5 (BsmBI cut no 3′ overhang) DNA in transcription buffer alone (U). Lanes 1–6: the same DNA transcribed with T7 polymerase for for 2 min (  1 ,   4 ), 5 min (  2 ,   5 ) and 20 min (  3 ,   6 ), then treated with RNase A, and deproteinized. In lanes 4–6 the DNA was preincubated for 20 min with TRF1 protein (text, Materials and Methods) prior to transcription. ( B ) pRST5 DNA (BsmBI cut, 54 nt sss 3′ overhang) was preincubated with 0, 2, 4 or 8 TRF2 monomers per terminal ss/ds junction, respectively, (lanes 1–4) prior to transcription for 30 min with T7 RNA polymerase, RNase treatment and deproteinization.
Figure Legend Snippet: Agarose gel electrophoretic analysis of DNA transcribed in the presence of TRF1 or TRF2. ( A ) Agarose gel electrophoresis of linear pRST5 (BsmBI cut no 3′ overhang) DNA in transcription buffer alone (U). Lanes 1–6: the same DNA transcribed with T7 polymerase for for 2 min ( 1 , 4 ), 5 min ( 2 , 5 ) and 20 min ( 3 , 6 ), then treated with RNase A, and deproteinized. In lanes 4–6 the DNA was preincubated for 20 min with TRF1 protein (text, Materials and Methods) prior to transcription. ( B ) pRST5 DNA (BsmBI cut, 54 nt sss 3′ overhang) was preincubated with 0, 2, 4 or 8 TRF2 monomers per terminal ss/ds junction, respectively, (lanes 1–4) prior to transcription for 30 min with T7 RNA polymerase, RNase treatment and deproteinization.

Techniques Used: Agarose Gel Electrophoresis

Architecture and composition of the telomeric tracts following transcription. ( A and B ) Linear pRST5 with no overhang or ( C–J ) a 54 nt 3′ overhang was transcribed for 30 min with T7 RNA polymerase in reactions containing (A and B) biotin-16-UTP or (C–J) UTP together with ATP and GTP (Materials and Methods). To detect any RNase resistant TERRA, samples were treated with RNase A and H followed by purification, and incubation with iron particles coated with streptavidin followed by removal of the unbound particles and EM preparation (text, Materials and Methods). The dense objects at the ( A ) loop junction or ( B ) junction of 3 DNAs are the iron particles. If RNase A treatment was not included, ( C and D ) linear molecules with loops at one end associated with a dense mass of RNA were present. ( E and F ): Following RNase A treatment, and purification, the loop junctions often contained a beaded particle. ( G and H ): Treatment of the samples with both RNase A and RNase H resulted in loop junctions, ( G ) some exhibiting a bead and ( H ) some not. ( I and J ) Loop junctions showing a short DNA stem extruded from the junction. Samples were mounted onto thin carbon supports followed by shadow casting with tungsten. Magnification bars are shown in each panel.
Figure Legend Snippet: Architecture and composition of the telomeric tracts following transcription. ( A and B ) Linear pRST5 with no overhang or ( C–J ) a 54 nt 3′ overhang was transcribed for 30 min with T7 RNA polymerase in reactions containing (A and B) biotin-16-UTP or (C–J) UTP together with ATP and GTP (Materials and Methods). To detect any RNase resistant TERRA, samples were treated with RNase A and H followed by purification, and incubation with iron particles coated with streptavidin followed by removal of the unbound particles and EM preparation (text, Materials and Methods). The dense objects at the ( A ) loop junction or ( B ) junction of 3 DNAs are the iron particles. If RNase A treatment was not included, ( C and D ) linear molecules with loops at one end associated with a dense mass of RNA were present. ( E and F ): Following RNase A treatment, and purification, the loop junctions often contained a beaded particle. ( G and H ): Treatment of the samples with both RNase A and RNase H resulted in loop junctions, ( G ) some exhibiting a bead and ( H ) some not. ( I and J ) Loop junctions showing a short DNA stem extruded from the junction. Samples were mounted onto thin carbon supports followed by shadow casting with tungsten. Magnification bars are shown in each panel.

Techniques Used: Purification, Incubation

EM visualization of transcription induced loops and HR products. ( A ) If the DNA templates were transcribed but not treated with RNAse A prior to preparation for EM, over 70% of the DNA had a bush of RNA attached at one end (see text for details). ( B–G ) With RNase A treatment, fields of linear molecules were present many of which contained a ( B ) tiny loop at one end and in the same fields examples of ( C ) several DNAs joined at one end were present, along with DNA bouquets containing (D) many molecules fused at one end. ( E ) When the samples above (B–G) were cleaved with NaeI that cuts 935 bp in from the telomeric end ∼1 kb fragments with loops (E) as well as bouquets with ∼1 kb arms (F) were observed. ( G ) Transcription of the minichromosome template shown in Figure 1D and processing as in B yielded DNAs with loops at both ends. Samples were prepared by surface spreading with cytochrome C (Materials and Methods) followed by rotary metal shadow casting. Bars indicating magnification are shown in each panel.
Figure Legend Snippet: EM visualization of transcription induced loops and HR products. ( A ) If the DNA templates were transcribed but not treated with RNAse A prior to preparation for EM, over 70% of the DNA had a bush of RNA attached at one end (see text for details). ( B–G ) With RNase A treatment, fields of linear molecules were present many of which contained a ( B ) tiny loop at one end and in the same fields examples of ( C ) several DNAs joined at one end were present, along with DNA bouquets containing (D) many molecules fused at one end. ( E ) When the samples above (B–G) were cleaved with NaeI that cuts 935 bp in from the telomeric end ∼1 kb fragments with loops (E) as well as bouquets with ∼1 kb arms (F) were observed. ( G ) Transcription of the minichromosome template shown in Figure 1D and processing as in B yielded DNAs with loops at both ends. Samples were prepared by surface spreading with cytochrome C (Materials and Methods) followed by rotary metal shadow casting. Bars indicating magnification are shown in each panel.

Techniques Used:

Agarose gel electrophoretic analysis of transcription mediated events. Agarose gel electrophoresis of linear pRST5 (BsmBI cut, no 3′ overhang) DNA in transcription buffer alone (U). If the DNA was transcribed for 30 min (Materials and Methods) and then deproteinized but not treated with RNase A, the DNA with RNA bound was present as a smear whether or not it was crosslinked with psoralen and UV ( 1 , 2 ). Following transcription, deproteinization and treatment with RNase A ( 3 ) the DNA was present as a ladder of bands with the monomer band shifted upward.
Figure Legend Snippet: Agarose gel electrophoretic analysis of transcription mediated events. Agarose gel electrophoresis of linear pRST5 (BsmBI cut, no 3′ overhang) DNA in transcription buffer alone (U). If the DNA was transcribed for 30 min (Materials and Methods) and then deproteinized but not treated with RNase A, the DNA with RNA bound was present as a smear whether or not it was crosslinked with psoralen and UV ( 1 , 2 ). Following transcription, deproteinization and treatment with RNase A ( 3 ) the DNA was present as a ladder of bands with the monomer band shifted upward.

Techniques Used: Agarose Gel Electrophoresis

27) Product Images from "Selective Export into Extracellular Vesicles and Function of tRNA Fragments during T Cell Activation"

Article Title: Selective Export into Extracellular Vesicles and Function of tRNA Fragments during T Cell Activation

Journal: Cell reports

doi: 10.1016/j.celrep.2018.11.073

EVs that Contain Intact Discrete RNA Species Are Separated from Protein Aggregates that Are Dominated by Fragmented RNAs (A) Schematic of a two-step purification procedure for separation of EVs from aggregates in cell culture supernatant. The supernatant was first subjected to differential centrifugation to remove live cells, dead cells, cell debris, and, finally, EVs, and aggregates were precipitated into 100,000 × g pellets. The 100,000 × g pellets were further separated by sucrose gradient into 6 fractions. (B) Western blot (top panel) analysis of sucrose gradient fractions of the separated 100,000 × g pellets and cell lysates prepared from the indicated numbers of cells in each lane. Bradford assay (bottom panel) determined total protein recovered in each fraction or cell lysate. * marks lanes containing similar concentrations of proteins from cell lysates (lane 2) and sucrose gradient fraction 3 (lane 7). (C) RNA 2100 Bioanalyzer analysis of large RNA species (left panel) and PAGE analysis of small RNA species ranging from 50 to 300 bp (right panel). Bottom panel shows total RNA yield from each fraction. * marks lanes with similar RNA yield from cells (lane 1) and sucrose gradient fraction 3 (lane 4). (D) qPCR analysis of miRNA abundance in equal volumes of RNA purified from each fraction. (E) qPCR analysis of the abundance of the indicated RNA species detected in fraction 3 (top) or in fraction 6 (bottom) left untreated or treated with RNase A or RNase A and Triton X-100. Data are representative of three independent experiments. Statistical significance is measured using a one-tailed t test: *p
Figure Legend Snippet: EVs that Contain Intact Discrete RNA Species Are Separated from Protein Aggregates that Are Dominated by Fragmented RNAs (A) Schematic of a two-step purification procedure for separation of EVs from aggregates in cell culture supernatant. The supernatant was first subjected to differential centrifugation to remove live cells, dead cells, cell debris, and, finally, EVs, and aggregates were precipitated into 100,000 × g pellets. The 100,000 × g pellets were further separated by sucrose gradient into 6 fractions. (B) Western blot (top panel) analysis of sucrose gradient fractions of the separated 100,000 × g pellets and cell lysates prepared from the indicated numbers of cells in each lane. Bradford assay (bottom panel) determined total protein recovered in each fraction or cell lysate. * marks lanes containing similar concentrations of proteins from cell lysates (lane 2) and sucrose gradient fraction 3 (lane 7). (C) RNA 2100 Bioanalyzer analysis of large RNA species (left panel) and PAGE analysis of small RNA species ranging from 50 to 300 bp (right panel). Bottom panel shows total RNA yield from each fraction. * marks lanes with similar RNA yield from cells (lane 1) and sucrose gradient fraction 3 (lane 4). (D) qPCR analysis of miRNA abundance in equal volumes of RNA purified from each fraction. (E) qPCR analysis of the abundance of the indicated RNA species detected in fraction 3 (top) or in fraction 6 (bottom) left untreated or treated with RNase A or RNase A and Triton X-100. Data are representative of three independent experiments. Statistical significance is measured using a one-tailed t test: *p

Techniques Used: Purification, Cell Culture, Centrifugation, Western Blot, Bradford Assay, Polyacrylamide Gel Electrophoresis, Real-time Polymerase Chain Reaction, One-tailed Test

Validation of EV Enrichment of tRFs and Their Responses to T Cell Activation (A) IGV visualization of tRFs of the indicated classes aligned to five representative mature tRNAs. (B) Electrophoretic analysis of products from the exponential phase of amplification in oligo(dT) RT-PCR assays for the indicated tRFs. We detected a band of the correct size and a larger product ~60 nt longer than 5′tRF or ~30 nt longer than 3′i-tRF, corresponding to amplification from full-length (FL) tRNAs. (C) Oligo(dT) qRT-PCR measurement of tRF abundance in EVs in resting (black bars) and stimulated (gray bars) conditions. (D) Oligo(dT) qRT-PCR analysis of the indicated RNA species detected in fraction 3 (F3) left untreated or treated with RNase A or with RNase A and Triton X-100. (E) tRF abundance in cells (black bars) and EVs (gray bars) under resting (R) and stimulated (S) conditions as determined by stem-loop qRT-PCR. Data are representative of at least three independent experiments. Statistical significance is measured using a one-tailed t test: *p
Figure Legend Snippet: Validation of EV Enrichment of tRFs and Their Responses to T Cell Activation (A) IGV visualization of tRFs of the indicated classes aligned to five representative mature tRNAs. (B) Electrophoretic analysis of products from the exponential phase of amplification in oligo(dT) RT-PCR assays for the indicated tRFs. We detected a band of the correct size and a larger product ~60 nt longer than 5′tRF or ~30 nt longer than 3′i-tRF, corresponding to amplification from full-length (FL) tRNAs. (C) Oligo(dT) qRT-PCR measurement of tRF abundance in EVs in resting (black bars) and stimulated (gray bars) conditions. (D) Oligo(dT) qRT-PCR analysis of the indicated RNA species detected in fraction 3 (F3) left untreated or treated with RNase A or with RNase A and Triton X-100. (E) tRF abundance in cells (black bars) and EVs (gray bars) under resting (R) and stimulated (S) conditions as determined by stem-loop qRT-PCR. Data are representative of at least three independent experiments. Statistical significance is measured using a one-tailed t test: *p

Techniques Used: Activation Assay, Amplification, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, One-tailed Test

28) Product Images from "Selective Export into Extracellular Vesicles and Function of tRNA Fragments during T Cell Activation"

Article Title: Selective Export into Extracellular Vesicles and Function of tRNA Fragments during T Cell Activation

Journal: Cell reports

doi: 10.1016/j.celrep.2018.11.073

EVs that Contain Intact Discrete RNA Species Are Separated from Protein Aggregates that Are Dominated by Fragmented RNAs (A) Schematic of a two-step purification procedure for separation of EVs from aggregates in cell culture supernatant. The supernatant was first subjected to differential centrifugation to remove live cells, dead cells, cell debris, and, finally, EVs, and aggregates were precipitated into 100,000 × g pellets. The 100,000 × g pellets were further separated by sucrose gradient into 6 fractions. (B) Western blot (top panel) analysis of sucrose gradient fractions of the separated 100,000 × g pellets and cell lysates prepared from the indicated numbers of cells in each lane. Bradford assay (bottom panel) determined total protein recovered in each fraction or cell lysate. * marks lanes containing similar concentrations of proteins from cell lysates (lane 2) and sucrose gradient fraction 3 (lane 7). (C) RNA 2100 Bioanalyzer analysis of large RNA species (left panel) and PAGE analysis of small RNA species ranging from 50 to 300 bp (right panel). Bottom panel shows total RNA yield from each fraction. * marks lanes with similar RNA yield from cells (lane 1) and sucrose gradient fraction 3 (lane 4). (D) qPCR analysis of miRNA abundance in equal volumes of RNA purified from each fraction. (E) qPCR analysis of the abundance of the indicated RNA species detected in fraction 3 (top) or in fraction 6 (bottom) left untreated or treated with RNase A or RNase A and Triton X-100. Data are representative of three independent experiments. Statistical significance is measured using a one-tailed t test: *p
Figure Legend Snippet: EVs that Contain Intact Discrete RNA Species Are Separated from Protein Aggregates that Are Dominated by Fragmented RNAs (A) Schematic of a two-step purification procedure for separation of EVs from aggregates in cell culture supernatant. The supernatant was first subjected to differential centrifugation to remove live cells, dead cells, cell debris, and, finally, EVs, and aggregates were precipitated into 100,000 × g pellets. The 100,000 × g pellets were further separated by sucrose gradient into 6 fractions. (B) Western blot (top panel) analysis of sucrose gradient fractions of the separated 100,000 × g pellets and cell lysates prepared from the indicated numbers of cells in each lane. Bradford assay (bottom panel) determined total protein recovered in each fraction or cell lysate. * marks lanes containing similar concentrations of proteins from cell lysates (lane 2) and sucrose gradient fraction 3 (lane 7). (C) RNA 2100 Bioanalyzer analysis of large RNA species (left panel) and PAGE analysis of small RNA species ranging from 50 to 300 bp (right panel). Bottom panel shows total RNA yield from each fraction. * marks lanes with similar RNA yield from cells (lane 1) and sucrose gradient fraction 3 (lane 4). (D) qPCR analysis of miRNA abundance in equal volumes of RNA purified from each fraction. (E) qPCR analysis of the abundance of the indicated RNA species detected in fraction 3 (top) or in fraction 6 (bottom) left untreated or treated with RNase A or RNase A and Triton X-100. Data are representative of three independent experiments. Statistical significance is measured using a one-tailed t test: *p

Techniques Used: Purification, Cell Culture, Centrifugation, Western Blot, Bradford Assay, Polyacrylamide Gel Electrophoresis, Real-time Polymerase Chain Reaction, One-tailed Test

Validation of EV Enrichment of tRFs and Their Responses to T Cell Activation (A) IGV visualization of tRFs of the indicated classes aligned to five representative mature tRNAs. (B) Electrophoretic analysis of products from the exponential phase of amplification in oligo(dT) RT-PCR assays for the indicated tRFs. We detected a band of the correct size and a larger product ~60 nt longer than 5′tRF or ~30 nt longer than 3′i-tRF, corresponding to amplification from full-length (FL) tRNAs. (C) Oligo(dT) qRT-PCR measurement of tRF abundance in EVs in resting (black bars) and stimulated (gray bars) conditions. (D) Oligo(dT) qRT-PCR analysis of the indicated RNA species detected in fraction 3 (F3) left untreated or treated with RNase A or with RNase A and Triton X-100. (E) tRF abundance in cells (black bars) and EVs (gray bars) under resting (R) and stimulated (S) conditions as determined by stem-loop qRT-PCR. Data are representative of at least three independent experiments. Statistical significance is measured using a one-tailed t test: *p
Figure Legend Snippet: Validation of EV Enrichment of tRFs and Their Responses to T Cell Activation (A) IGV visualization of tRFs of the indicated classes aligned to five representative mature tRNAs. (B) Electrophoretic analysis of products from the exponential phase of amplification in oligo(dT) RT-PCR assays for the indicated tRFs. We detected a band of the correct size and a larger product ~60 nt longer than 5′tRF or ~30 nt longer than 3′i-tRF, corresponding to amplification from full-length (FL) tRNAs. (C) Oligo(dT) qRT-PCR measurement of tRF abundance in EVs in resting (black bars) and stimulated (gray bars) conditions. (D) Oligo(dT) qRT-PCR analysis of the indicated RNA species detected in fraction 3 (F3) left untreated or treated with RNase A or with RNase A and Triton X-100. (E) tRF abundance in cells (black bars) and EVs (gray bars) under resting (R) and stimulated (S) conditions as determined by stem-loop qRT-PCR. Data are representative of at least three independent experiments. Statistical significance is measured using a one-tailed t test: *p

Techniques Used: Activation Assay, Amplification, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, One-tailed Test

29) Product Images from "Optimization of ribosome profiling using low-input brain tissue from fragile X syndrome model mice"

Article Title: Optimization of ribosome profiling using low-input brain tissue from fragile X syndrome model mice

Journal: Nucleic Acids Research

doi: 10.1093/nar/gky1292

RPF GC content is RNase-species independent. ( A ) 3.8 A 260 homogenate from hippocampi of one P35 male mouse was digested with 100ng RNase A (Sigma, # R4875) + 60U RNase T1 (Thermo Fisher Scientific, #EN0542)/ A 260 , at 25°C for 30min and applied to a 10–50% (w/v) sucrose gradient. ( B ) 3.8 A 260 homogenate from hippocampi of one P35 mouse was digested with 5U RNase I (Ambion, #AM2294)/ A 260 , at 25°C for 45min and applied to a 10–50% (w/v) sucrose gradient. ( C ) Nucleotide composition at each position of RPFs mapped to CDS from ribosomes in (A). ( D ) Nucleotide composition at each position of RPFs mapped to CDS from ribosomes in (B). ( E ) Nucleotide composition at each position of RPFs mapped to CDS from mouse embryonic stem cells (mESCs) (data from Ingolia et al. ) ( 16 ). A 600 μl aliquot of lysate was treated with 15 μl RNase I at 100 U/μl for 45 min at 25°C. ( F ) Nucleotide composition at each position of RPFs mapped to CDS from human embryonic stem cell (hESC)-derived neurons (data from Grabole et al. ) ( 42 ). 5 U TruSeq Ribo Profile Nuclease/ A 260 at 25°C for 45 min.
Figure Legend Snippet: RPF GC content is RNase-species independent. ( A ) 3.8 A 260 homogenate from hippocampi of one P35 male mouse was digested with 100ng RNase A (Sigma, # R4875) + 60U RNase T1 (Thermo Fisher Scientific, #EN0542)/ A 260 , at 25°C for 30min and applied to a 10–50% (w/v) sucrose gradient. ( B ) 3.8 A 260 homogenate from hippocampi of one P35 mouse was digested with 5U RNase I (Ambion, #AM2294)/ A 260 , at 25°C for 45min and applied to a 10–50% (w/v) sucrose gradient. ( C ) Nucleotide composition at each position of RPFs mapped to CDS from ribosomes in (A). ( D ) Nucleotide composition at each position of RPFs mapped to CDS from ribosomes in (B). ( E ) Nucleotide composition at each position of RPFs mapped to CDS from mouse embryonic stem cells (mESCs) (data from Ingolia et al. ) ( 16 ). A 600 μl aliquot of lysate was treated with 15 μl RNase I at 100 U/μl for 45 min at 25°C. ( F ) Nucleotide composition at each position of RPFs mapped to CDS from human embryonic stem cell (hESC)-derived neurons (data from Grabole et al. ) ( 42 ). 5 U TruSeq Ribo Profile Nuclease/ A 260 at 25°C for 45 min.

Techniques Used: Derivative Assay

RPF GC content and length depend on the RNase digestion protocol. ( A ) Lysates from human iPSC neuron samples spanning a wide range of amounts were digested with 100 ng RNase A + 60U RNase T1/ A 260 at 25°C for 30 min. Monosomal RNA was extracted from monosomal fractions of sucrose gradients and quantified with Nanodrop. GC contents were calculated as in Figure 2A and the peaks of length distributions of RPFs mapped to CDS were also determined. Scatter plots with Pearson correlation coefficients show the negative correlation between 80S monosomal RNA amounts (log 2 scale) and the GC contents (black) or RPF lengths (red). ( B ) Lysates from human iPSC samples were digested with 20 ng RNase A + 12 U RNase T1/ A 260 at 25°C for 30 min. Scatter plots with Pearson correlation coefficients show the negative correlation between 80S monosomal RNA amounts (log 2 scale) and the GC contents (black) or RPF lengths (red). ( C ) Nucleotide composition at each position of RPFs mapped to CDS from mESC-derived neurons with an alternative protocol of RNase digestion (data from Zappulo et al. ) ( 43 ). 70 U RNase I at 25°C for 40 min.
Figure Legend Snippet: RPF GC content and length depend on the RNase digestion protocol. ( A ) Lysates from human iPSC neuron samples spanning a wide range of amounts were digested with 100 ng RNase A + 60U RNase T1/ A 260 at 25°C for 30 min. Monosomal RNA was extracted from monosomal fractions of sucrose gradients and quantified with Nanodrop. GC contents were calculated as in Figure 2A and the peaks of length distributions of RPFs mapped to CDS were also determined. Scatter plots with Pearson correlation coefficients show the negative correlation between 80S monosomal RNA amounts (log 2 scale) and the GC contents (black) or RPF lengths (red). ( B ) Lysates from human iPSC samples were digested with 20 ng RNase A + 12 U RNase T1/ A 260 at 25°C for 30 min. Scatter plots with Pearson correlation coefficients show the negative correlation between 80S monosomal RNA amounts (log 2 scale) and the GC contents (black) or RPF lengths (red). ( C ) Nucleotide composition at each position of RPFs mapped to CDS from mESC-derived neurons with an alternative protocol of RNase digestion (data from Zappulo et al. ) ( 43 ). 70 U RNase I at 25°C for 40 min.

Techniques Used: Derivative Assay

The GC-content correlated batch effects are caused by incomplete RNase digestion. ( A ) Hippocampi from one P35 WT mouse were homogenized and the homogenate was aliquoted for the titration experiment. 0.5 unit A 260 homogenate containing 2 μg RNA (measured with Qubit HS RNA kit) in 0.3 ml volume was used for digestion at each RNase concentration. Digested homogenates were separated on 10–50% (w/v) sucrose gradients. Profile of hippocampal ribosomes after the digestion at the lowest concentration1 [Conc.1, 4.8ng RNase A (Ambion, #AM2270) + 0.6 U RNase T1 (Thermo Fisher Scientific, #EN0542)/μg RNA × 2 μg RNA in 0.3 ml at 25°C for 30 min] and sucrose gradient fractionation. ( B ) Profile of hippocampal ribosomes after the digestion at the concentration2 (Conc.2, 24 ng RNase A + 3U RNase T1/μg RNA × 2 μg RNA in 0.3 ml at 25°C for 30 min) and sucrose gradient fractionation. ( C ) Profile of hippocampal ribosomes after the digestion at the concentration3 (Conc.3, 120 ng RNase A + 15 U RNase T1/μg RNA × 2 μg RNA in 0.3 ml at 25°C for 30 min) and sucrose gradient fractionation. ( D ) Profile of hippocampal ribosomes after the digestion at the concentration4 (Conc.4, 600 ng RNase A + 75 U RNase T1/μg RNA × 2 μg RNA in 0.3 ml at 25°C for 30 min) and sucrose gradient fractionation. ( E ) Profile of hippocampal ribosomes after the digestion at the highest concentration5 (Conc.5, 3000 ng RNase A + 375 U RNase T1/μg RNA × 2 μg RNA RNA in 0.3 ml at 25°C for 30 min) and sucrose gradient fractionation. ( F ) Scatter plots with Pearson correlation coefficients show the negative correlation between RNase concentrations (log 5 scale) and the GC contents (black) or RPF lengths (red).
Figure Legend Snippet: The GC-content correlated batch effects are caused by incomplete RNase digestion. ( A ) Hippocampi from one P35 WT mouse were homogenized and the homogenate was aliquoted for the titration experiment. 0.5 unit A 260 homogenate containing 2 μg RNA (measured with Qubit HS RNA kit) in 0.3 ml volume was used for digestion at each RNase concentration. Digested homogenates were separated on 10–50% (w/v) sucrose gradients. Profile of hippocampal ribosomes after the digestion at the lowest concentration1 [Conc.1, 4.8ng RNase A (Ambion, #AM2270) + 0.6 U RNase T1 (Thermo Fisher Scientific, #EN0542)/μg RNA × 2 μg RNA in 0.3 ml at 25°C for 30 min] and sucrose gradient fractionation. ( B ) Profile of hippocampal ribosomes after the digestion at the concentration2 (Conc.2, 24 ng RNase A + 3U RNase T1/μg RNA × 2 μg RNA in 0.3 ml at 25°C for 30 min) and sucrose gradient fractionation. ( C ) Profile of hippocampal ribosomes after the digestion at the concentration3 (Conc.3, 120 ng RNase A + 15 U RNase T1/μg RNA × 2 μg RNA in 0.3 ml at 25°C for 30 min) and sucrose gradient fractionation. ( D ) Profile of hippocampal ribosomes after the digestion at the concentration4 (Conc.4, 600 ng RNase A + 75 U RNase T1/μg RNA × 2 μg RNA in 0.3 ml at 25°C for 30 min) and sucrose gradient fractionation. ( E ) Profile of hippocampal ribosomes after the digestion at the highest concentration5 (Conc.5, 3000 ng RNase A + 375 U RNase T1/μg RNA × 2 μg RNA RNA in 0.3 ml at 25°C for 30 min) and sucrose gradient fractionation. ( F ) Scatter plots with Pearson correlation coefficients show the negative correlation between RNase concentrations (log 5 scale) and the GC contents (black) or RPF lengths (red).

Techniques Used: Titration, Concentration Assay, Fractionation

30) Product Images from "Kaposi's Sarcoma-Associated Herpesvirus K8 Is an RNA Binding Protein That Regulates Viral DNA Replication in Coordination with a Noncoding RNA"

Article Title: Kaposi's Sarcoma-Associated Herpesvirus K8 Is an RNA Binding Protein That Regulates Viral DNA Replication in Coordination with a Noncoding RNA

Journal: Journal of Virology

doi: 10.1128/JVI.02177-17

Validation of K8 RNA binding property and identification of K8-associated RNAs using a CLIP-seq approach. (A) TPA-induced BCBL-1 cells were UV irradiated and subjected to immunoprecipitation with K8 antibody. Different amounts of RNase A (++, 2 μg/ml; +, 1 μg/ml) were added to the washing buffer during immunoprecipitation. Coprecipitated RNA was labeled with biotin at the 3′ end and spotted onto a nylon membrane. The biotinylated RNA was then analyzed with a chemiluminescent nucleic acid detection module kit. (B) Schematic illustration of the CLIP-seq procedure. TPA-induced BCBL-1 cells were UV irradiated and immunoprecipitated with K8 antibody. Different amounts of RNase A were added to the washing buffer. The coprecipitated RNAs were phosphorylated at the 5′ end with T4 polynucleotide kinase (PNK) and dephosphorylated with alkaline phosphatase at the 3′ end. The RNAs were ligated to the 3′ linker and then to the 5′ linker labeled with γ- 32 P. The RNA-protein complex was resolved in SDS-PAGE, and RNAs were isolated and amplified by RT-PCR. The PCR products were analyzed by high-throughput sequencing. (C) Autoradiogram of γ- 32 P-labeled RNA cross-linked to K8. Immunoprecipitation was performed with anti-K8 or anti-ORF45 antibody. (Bottom) Input of K8 and β-actin. (D) RT-PCR products from K8-CLIP. (E) Experimental design of CLIP-seq. For each set of CLIP experiments (IgG, KSHV-negative [BJAB], KSHV1 [BCBL-1], and KSHV2 [BCBL-1]), two biological repeats were carried out. Each biological repeat contained three technical repeats. (F) Library statistics of CLIP-seq. Two biological repeats from K8-CLIP-seq (KSHV1 and KSHV2) contained 67,953,806 and 81,632,254 clean reads, respectively. In contrast, there were only 402,896 and 3,472,238 clean reads in the IgG control and KSHV negative (BJAB) data sets, respectively. (G) Libraries from two biological repeats were highly similar. The correlation coefficient between two biological repeats (KSHV1 and KSHV2) was above 0.97.
Figure Legend Snippet: Validation of K8 RNA binding property and identification of K8-associated RNAs using a CLIP-seq approach. (A) TPA-induced BCBL-1 cells were UV irradiated and subjected to immunoprecipitation with K8 antibody. Different amounts of RNase A (++, 2 μg/ml; +, 1 μg/ml) were added to the washing buffer during immunoprecipitation. Coprecipitated RNA was labeled with biotin at the 3′ end and spotted onto a nylon membrane. The biotinylated RNA was then analyzed with a chemiluminescent nucleic acid detection module kit. (B) Schematic illustration of the CLIP-seq procedure. TPA-induced BCBL-1 cells were UV irradiated and immunoprecipitated with K8 antibody. Different amounts of RNase A were added to the washing buffer. The coprecipitated RNAs were phosphorylated at the 5′ end with T4 polynucleotide kinase (PNK) and dephosphorylated with alkaline phosphatase at the 3′ end. The RNAs were ligated to the 3′ linker and then to the 5′ linker labeled with γ- 32 P. The RNA-protein complex was resolved in SDS-PAGE, and RNAs were isolated and amplified by RT-PCR. The PCR products were analyzed by high-throughput sequencing. (C) Autoradiogram of γ- 32 P-labeled RNA cross-linked to K8. Immunoprecipitation was performed with anti-K8 or anti-ORF45 antibody. (Bottom) Input of K8 and β-actin. (D) RT-PCR products from K8-CLIP. (E) Experimental design of CLIP-seq. For each set of CLIP experiments (IgG, KSHV-negative [BJAB], KSHV1 [BCBL-1], and KSHV2 [BCBL-1]), two biological repeats were carried out. Each biological repeat contained three technical repeats. (F) Library statistics of CLIP-seq. Two biological repeats from K8-CLIP-seq (KSHV1 and KSHV2) contained 67,953,806 and 81,632,254 clean reads, respectively. In contrast, there were only 402,896 and 3,472,238 clean reads in the IgG control and KSHV negative (BJAB) data sets, respectively. (G) Libraries from two biological repeats were highly similar. The correlation coefficient between two biological repeats (KSHV1 and KSHV2) was above 0.97.

Techniques Used: RNA Binding Assay, Cross-linking Immunoprecipitation, Irradiation, Immunoprecipitation, Labeling, SDS Page, Isolation, Amplification, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Next-Generation Sequencing

Association of K8 with ori-Lyt DNA is mediated by RNA. (A) Schematic illustration of the KSHV ori-Lyt core domain and DNA fragments that were used in the DNA affinity assay. (B) Three biotinylated ori-Lyt DNA fragments and an irrelevant DNA fragment from the ORF45 coding region as a control were prepared by PCR, conjugated on magnetic beads, and incubated with TPA-induced BCBL-1 nuclear extract with and without treatment with RNase A. After washing, samples were assayed by Western blotting with antibodies as indicated. (C) Binding of K8 to ori-Lyt DNA was determined in BAC16 (BAC WT) and BAC-K8GDDGR by ChIP assay with anti-K8 antibody. The positions of the amplicons (3.1F and 12F) are shown in panel A. The error bars indicate SD.
Figure Legend Snippet: Association of K8 with ori-Lyt DNA is mediated by RNA. (A) Schematic illustration of the KSHV ori-Lyt core domain and DNA fragments that were used in the DNA affinity assay. (B) Three biotinylated ori-Lyt DNA fragments and an irrelevant DNA fragment from the ORF45 coding region as a control were prepared by PCR, conjugated on magnetic beads, and incubated with TPA-induced BCBL-1 nuclear extract with and without treatment with RNase A. After washing, samples were assayed by Western blotting with antibodies as indicated. (C) Binding of K8 to ori-Lyt DNA was determined in BAC16 (BAC WT) and BAC-K8GDDGR by ChIP assay with anti-K8 antibody. The positions of the amplicons (3.1F and 12F) are shown in panel A. The error bars indicate SD.

Techniques Used: Polymerase Chain Reaction, Magnetic Beads, Incubation, Western Blot, Binding Assay, BAC Assay, Chromatin Immunoprecipitation

T1.4 RNA associated with ori-Lyt DNA. (A) Alignment of T1.4 RNA with ori-Lyt DNA sequences in the KSHV genome revealing potential base pairing between them. (B) Schematic illustration of the ChIRP assay procedure. Glutaraldehyde was applied to induce DNA-RNA cross-linking. Chromatin was sonicated and then incubated with biotinylated oligonucleotide probe specifically to the target RNA. Streptavidin beads were added to pull down the biotinylated RNA and bound DNA. qPCR was used to detect the ori-Lyt DNA. (C) Validation of the efficiency of T1.4 probe in pulldown of T1.4 RNA. A ChIRP assay was performed using probes targeting T1.4 or LacZ. Precipitated RNAs were purified and subjected to RT-qPCR with primer against T1.4. (D) T1.4 RNA associates with ori-Lyt DNA. Two sets of probes were used to perform ChIRP experiments. RNase H, RNase A, or trypsin was added in washing buffer during the experiments to digest the RNA or protein. The pulled down DNA was amplified by primers for the 12F region of ori-Lyt. The error bars indicate SD.
Figure Legend Snippet: T1.4 RNA associated with ori-Lyt DNA. (A) Alignment of T1.4 RNA with ori-Lyt DNA sequences in the KSHV genome revealing potential base pairing between them. (B) Schematic illustration of the ChIRP assay procedure. Glutaraldehyde was applied to induce DNA-RNA cross-linking. Chromatin was sonicated and then incubated with biotinylated oligonucleotide probe specifically to the target RNA. Streptavidin beads were added to pull down the biotinylated RNA and bound DNA. qPCR was used to detect the ori-Lyt DNA. (C) Validation of the efficiency of T1.4 probe in pulldown of T1.4 RNA. A ChIRP assay was performed using probes targeting T1.4 or LacZ. Precipitated RNAs were purified and subjected to RT-qPCR with primer against T1.4. (D) T1.4 RNA associates with ori-Lyt DNA. Two sets of probes were used to perform ChIRP experiments. RNase H, RNase A, or trypsin was added in washing buffer during the experiments to digest the RNA or protein. The pulled down DNA was amplified by primers for the 12F region of ori-Lyt. The error bars indicate SD.

Techniques Used: Sonication, Incubation, Real-time Polymerase Chain Reaction, Purification, Quantitative RT-PCR, Amplification

31) Product Images from "The wild-type Schizosaccharomyces pombe mat1 imprint consists of two ribonucleotides"

Article Title: The wild-type Schizosaccharomyces pombe mat1 imprint consists of two ribonucleotides

Journal: EMBO Reports

doi: 10.1038/sj.embor.7400576

Escherichia coli  DNA ligase, but not T4 DNA ligase, can be used to detect a 3′-terminal ribonucleotide on a DNA fragment. ( A ) NaOH or Rnase. A hydrolysis of a ribonucleotide phosphodiester bond results in the formation of equimolar amounts of 2′- and 3′-terminal phosphates and a 5′ hydroxyl. The 2′ and 3′ positions of the ribose are marked. The deoxyribonucleotide, following the ribonucleotide in the DNA–RNA–DNA chain, is depicted in grey. The intermediate 2′–3′ cyclic phosphate is shown.
Figure Legend Snippet: Escherichia coli DNA ligase, but not T4 DNA ligase, can be used to detect a 3′-terminal ribonucleotide on a DNA fragment. ( A ) NaOH or Rnase. A hydrolysis of a ribonucleotide phosphodiester bond results in the formation of equimolar amounts of 2′- and 3′-terminal phosphates and a 5′ hydroxyl. The 2′ and 3′ positions of the ribose are marked. The deoxyribonucleotide, following the ribonucleotide in the DNA–RNA–DNA chain, is depicted in grey. The intermediate 2′–3′ cyclic phosphate is shown.

Techniques Used:

Detection of a 3′-terminal ribonucleotide in the 5′ fragment at the hydrolysed mat1 imprint. ( A ) Line drawing of the experimental strategy, using ligation-mediated PCR (LM-PCR) and RNase A digestion. Chromosomal and linker DNA are shown by thick and thin black lines, respectively. The ribonucleotide is indicated by a circle. The positions of the primers, used for LM-PCR and control PCR, are given as black and grey arrows, respectively. ( B,C ) LM-PCR of the 3′ end at the imprint using two different linkers (part of the given sequences). Linkers (grey boxes) were designed to allow ligation to nicked ( B ; primer LM1/LM2) or gapped ( C ; primer LM1A/LM2) molecules. The control amplification of a flanking sequence is shown. The number of cycles used in the PCR reaction is shown above each lane.
Figure Legend Snippet: Detection of a 3′-terminal ribonucleotide in the 5′ fragment at the hydrolysed mat1 imprint. ( A ) Line drawing of the experimental strategy, using ligation-mediated PCR (LM-PCR) and RNase A digestion. Chromosomal and linker DNA are shown by thick and thin black lines, respectively. The ribonucleotide is indicated by a circle. The positions of the primers, used for LM-PCR and control PCR, are given as black and grey arrows, respectively. ( B,C ) LM-PCR of the 3′ end at the imprint using two different linkers (part of the given sequences). Linkers (grey boxes) were designed to allow ligation to nicked ( B ; primer LM1/LM2) or gapped ( C ; primer LM1A/LM2) molecules. The control amplification of a flanking sequence is shown. The number of cycles used in the PCR reaction is shown above each lane.

Techniques Used: Ligation, Polymerase Chain Reaction, Amplification, Sequencing

( B ) A DNA linker can be efficiently ligated to a terminal ribonucleotide with 2′ or 3′ phosphate using T4, but not E. coli, DNA ligase, forming products resistant to re-hydrolysis. Ligation using T4 and E. coli ). Radioactive 5′-end label is shown with an asterisk. The ribonucleotide is indicated by a circle. For both panels: lane 1, end-labelled oligonucleotide ART26; lanes 2–5, experiments using ART26 hydrolysed with NaOH as a substrate (N); lanes 6–9, experiments using ART26 hydrolysed with RNase A (R); lanes 2,6, hydrolysed ART26 substrate; lanes 3,7, linker ligation products; lanes 4,8, re-hydrolysis of the ligation product with NaOH; lanes 5,9, re-hydrolysis of the ligation product with RNase A. The fragment, marked IV (lane 3), formed from ligation of the linker to the unhydrolysed oligonucleotide I serves as an internal control; following NaOH or RNase A treatment (lanes 4,5), the band disappears, showing that the RNA bond with a hydroxyl at the 2′ position is efficiently hydrolysed under the conditions of the experiment. ( C ) A DNA oligonucleotide containing a 3′-terminal ribonucleotide devoid of 2′ or 3′ phosphate is a substrate for E. coli DNA ligase, and the ligation product can be re-hydrolysed using RNase A. Lane 1, substrate ART26 hydrolysed with NaOH, dephosphorylated with Antarctic phosphatase and 5′-end labelled; lane 2, ligation products; lane 3, ligation products re-hydrolysed with RNase A. An outline of the experiment is given below in a line drawing (description as in B ).
Figure Legend Snippet: ( B ) A DNA linker can be efficiently ligated to a terminal ribonucleotide with 2′ or 3′ phosphate using T4, but not E. coli, DNA ligase, forming products resistant to re-hydrolysis. Ligation using T4 and E. coli ). Radioactive 5′-end label is shown with an asterisk. The ribonucleotide is indicated by a circle. For both panels: lane 1, end-labelled oligonucleotide ART26; lanes 2–5, experiments using ART26 hydrolysed with NaOH as a substrate (N); lanes 6–9, experiments using ART26 hydrolysed with RNase A (R); lanes 2,6, hydrolysed ART26 substrate; lanes 3,7, linker ligation products; lanes 4,8, re-hydrolysis of the ligation product with NaOH; lanes 5,9, re-hydrolysis of the ligation product with RNase A. The fragment, marked IV (lane 3), formed from ligation of the linker to the unhydrolysed oligonucleotide I serves as an internal control; following NaOH or RNase A treatment (lanes 4,5), the band disappears, showing that the RNA bond with a hydroxyl at the 2′ position is efficiently hydrolysed under the conditions of the experiment. ( C ) A DNA oligonucleotide containing a 3′-terminal ribonucleotide devoid of 2′ or 3′ phosphate is a substrate for E. coli DNA ligase, and the ligation product can be re-hydrolysed using RNase A. Lane 1, substrate ART26 hydrolysed with NaOH, dephosphorylated with Antarctic phosphatase and 5′-end labelled; lane 2, ligation products; lane 3, ligation products re-hydrolysed with RNase A. An outline of the experiment is given below in a line drawing (description as in B ).

Techniques Used: Ligation

32) Product Images from "Noncoding RNA Ginir functions as an oncogene by associating with centrosomal proteins"

Article Title: Noncoding RNA Ginir functions as an oncogene by associating with centrosomal proteins

Journal: PLoS Biology

doi: 10.1371/journal.pbio.2004204

Ginir RNA impairs interaction between Cep112 and Brca1 proteins leading to genomic instability. (A) RNA pull-down with biotinylated Ginir RNA in NIH/3T3 cells followed by western blotting with Brca1 antibody (sc-646, Santa Cruz). Pull-down with unbiotinylated RNA probe served as control for nonspecific binding. (B-D) RIP performed using both Cep112 and Brca1 antibodies followed by RNA isolation and RT-PCR with Ginir specific primers (G2F-G2R) in NIH/3T3-GinirA (B) and NIH/3T3-GinirB (C) cells. RIP assay was also followed by RT-PCR using nonspecific primers for U6 snRNA (D). Anti-IgG IP served as control for nonspecific interaction. (E) In silico model of Cep112 and Brca1 interaction generated through computational docking using ZDOCK tool. (F and G) Co-IP of Cep112 and Brca1 proteins in NIH/3T3-EV cells. IP was performed with Brca1 antibody (sc-646, Santa Cruz) followed by immunoblotting with Cep112 antibody (sc-246162, Santa Cruz) (F) and vice versa (G). Anti-IgG IP served as a control for nonspecific binding to the antibody. (H and I) Western blotting with Cep112 antibody (24928-1-AP, Proteintech) for validation of Flag-Cep112 overexpression in NIH/3T3 cells (H). Fifty μg of whole-cell protein lysates from each of the mentioned cell lines was loaded on 7% SDS-PAGE. Tubulin served as internal loading control (I). (J) Co-IP of Brca1 and Flag-Cep112 in NIH-Flag-Cep112 cells. IP was performed with Flag1 antibody followed by immunoblotting with Brca1 antibody (20649-1-AP, Proteintech). Anti-IgG IP served as a control for nonspecific binding to the antibody. (K) Co-IP of Brca1 and Cep112 proteins in NIH/3T3-EV, NIH/3T3-Ginir, and NIH-Ginir-shGinir2 cells. IP was performed with Brca1 antibody (sc-646, Santa Cruz) followed by immunoblotting with Cep112 antibody (24928-1-AP, Proteintech). Anti-IgG IP served as a control for nonspecific binding to the antibody. (L) Confocal images showing colocalisation of Brca1 with γ-tubulin in NIH/3T3-EV cell line. Nuclei were stained with DAPI. Scale bars, 10 μm. (M) Confocal images showing colocalisation of Brca1 protein with Cep112 protein in NIH/3T3-EV cell line. Nuclei were stained with DAPI. Scale bars, 20 μm. (N) Confocal imaging for Brca1 expression in NIH/3T3-EV and NIH/3T3-Ginir cells. Scale bars, 20 μm. (O) RNA-FISH using Ginir-specific probe (probe 1, FAM labelled) in NIH/3T3-EV cells visualised by confocal imaging. Scale bars, 20 μm. (P) Co-IP of Brca1 and Cep112 in NIH/3T3-Ginir(C) cells wherein lysates were treated independently with RNase (A, H, and III mix) or RNasin. Both RNase- and RNasin-treated lysates were immunoprecipitated with Brca1 antibody (sc-646, Santa Cruz) and blotted using Cep112 antibody (sc-246163, Santa Cruz). IP with anti-IgG served as control. Brca1, breast cancer type 1 susceptibility protein; Cep112, centrosomal protein 112; FAM, fluorescein amidite; Ginir, Genomic Instability Inducing RNA; IgG, immunoglobulin G; IP, immunoprecipitation; RIP, RNA-immunoprecipitation; RNasin, RNase inhibitor; RT-PCR, reverse transcription polymerase chain reaction; snRNA, small nuclear RNA.
Figure Legend Snippet: Ginir RNA impairs interaction between Cep112 and Brca1 proteins leading to genomic instability. (A) RNA pull-down with biotinylated Ginir RNA in NIH/3T3 cells followed by western blotting with Brca1 antibody (sc-646, Santa Cruz). Pull-down with unbiotinylated RNA probe served as control for nonspecific binding. (B-D) RIP performed using both Cep112 and Brca1 antibodies followed by RNA isolation and RT-PCR with Ginir specific primers (G2F-G2R) in NIH/3T3-GinirA (B) and NIH/3T3-GinirB (C) cells. RIP assay was also followed by RT-PCR using nonspecific primers for U6 snRNA (D). Anti-IgG IP served as control for nonspecific interaction. (E) In silico model of Cep112 and Brca1 interaction generated through computational docking using ZDOCK tool. (F and G) Co-IP of Cep112 and Brca1 proteins in NIH/3T3-EV cells. IP was performed with Brca1 antibody (sc-646, Santa Cruz) followed by immunoblotting with Cep112 antibody (sc-246162, Santa Cruz) (F) and vice versa (G). Anti-IgG IP served as a control for nonspecific binding to the antibody. (H and I) Western blotting with Cep112 antibody (24928-1-AP, Proteintech) for validation of Flag-Cep112 overexpression in NIH/3T3 cells (H). Fifty μg of whole-cell protein lysates from each of the mentioned cell lines was loaded on 7% SDS-PAGE. Tubulin served as internal loading control (I). (J) Co-IP of Brca1 and Flag-Cep112 in NIH-Flag-Cep112 cells. IP was performed with Flag1 antibody followed by immunoblotting with Brca1 antibody (20649-1-AP, Proteintech). Anti-IgG IP served as a control for nonspecific binding to the antibody. (K) Co-IP of Brca1 and Cep112 proteins in NIH/3T3-EV, NIH/3T3-Ginir, and NIH-Ginir-shGinir2 cells. IP was performed with Brca1 antibody (sc-646, Santa Cruz) followed by immunoblotting with Cep112 antibody (24928-1-AP, Proteintech). Anti-IgG IP served as a control for nonspecific binding to the antibody. (L) Confocal images showing colocalisation of Brca1 with γ-tubulin in NIH/3T3-EV cell line. Nuclei were stained with DAPI. Scale bars, 10 μm. (M) Confocal images showing colocalisation of Brca1 protein with Cep112 protein in NIH/3T3-EV cell line. Nuclei were stained with DAPI. Scale bars, 20 μm. (N) Confocal imaging for Brca1 expression in NIH/3T3-EV and NIH/3T3-Ginir cells. Scale bars, 20 μm. (O) RNA-FISH using Ginir-specific probe (probe 1, FAM labelled) in NIH/3T3-EV cells visualised by confocal imaging. Scale bars, 20 μm. (P) Co-IP of Brca1 and Cep112 in NIH/3T3-Ginir(C) cells wherein lysates were treated independently with RNase (A, H, and III mix) or RNasin. Both RNase- and RNasin-treated lysates were immunoprecipitated with Brca1 antibody (sc-646, Santa Cruz) and blotted using Cep112 antibody (sc-246163, Santa Cruz). IP with anti-IgG served as control. Brca1, breast cancer type 1 susceptibility protein; Cep112, centrosomal protein 112; FAM, fluorescein amidite; Ginir, Genomic Instability Inducing RNA; IgG, immunoglobulin G; IP, immunoprecipitation; RIP, RNA-immunoprecipitation; RNasin, RNase inhibitor; RT-PCR, reverse transcription polymerase chain reaction; snRNA, small nuclear RNA.

Techniques Used: Western Blot, Binding Assay, Isolation, Reverse Transcription Polymerase Chain Reaction, In Silico, Generated, Co-Immunoprecipitation Assay, Over Expression, SDS Page, Staining, Imaging, Expressing, Fluorescence In Situ Hybridization, Immunoprecipitation

Expression of Ginir/Giniras transcripts during mouse embryonic development and in adult tissues. (A) List of several ESTs spanning Ginir sequence and showing significant similarity to Ginir ( http://blast.ncbi.nlm.nih.gov/Blast.cgi ). (B) RPA of RNA isolated from NIH/3T3 cells using PCR-generated sense or antisense riboprobes specific to Ginir sequence. Yeast total RNA served as control for RNase A/T1 activity. (C and D) Expression of Ginir/Giniras RNA in different stages of development (dpc) of mouse embryos (C) and in tissues from different organs of adult mice (D) using strand-specific cDNA synthesis and RT-PCR with G1F-G1R primers. Gapdh served as internal loading control. (E) Whole-mount ISH using LNA probes for Ginir (FAM labelled, green) or Giniras (TexRed labelled, red) on 10.5 dpc mouse embryos showing differential expression in brain (‘br’), forelimbs (‘fl’), and spinal cord (‘sc’). Whole-mount embryos treated with RNase A served as negative control for fluorescence. (F) FISH using LNA probes for Ginir (Green) or Giniras (Red) on embryo sections of 13.5 and 14.5 dpc embryos showing differential expression in forebrain (‘fb’), midbrain (‘mb’), hypothalamus (‘ht’), and limbs (‘li’). Embryo sections treated with RNase A served as a negative control. dpc, days post coitum; EST, expressed sequence tag; FAM, fluorescein amidite; FISH, fluorescence in situ hybridisation; Gapdh, glyceride 3-phosphate dehydrogenase; Ginir, Genomic Instability Inducing RNA; Giniras, antisense RNA of Ginir; ISH, in situ hybridisation; LNA, locked nucleic acid; PCR, polymerase chain reaction; RPA, ribonuclease protection assay; RT-PCR, reverse transcription PCR.
Figure Legend Snippet: Expression of Ginir/Giniras transcripts during mouse embryonic development and in adult tissues. (A) List of several ESTs spanning Ginir sequence and showing significant similarity to Ginir ( http://blast.ncbi.nlm.nih.gov/Blast.cgi ). (B) RPA of RNA isolated from NIH/3T3 cells using PCR-generated sense or antisense riboprobes specific to Ginir sequence. Yeast total RNA served as control for RNase A/T1 activity. (C and D) Expression of Ginir/Giniras RNA in different stages of development (dpc) of mouse embryos (C) and in tissues from different organs of adult mice (D) using strand-specific cDNA synthesis and RT-PCR with G1F-G1R primers. Gapdh served as internal loading control. (E) Whole-mount ISH using LNA probes for Ginir (FAM labelled, green) or Giniras (TexRed labelled, red) on 10.5 dpc mouse embryos showing differential expression in brain (‘br’), forelimbs (‘fl’), and spinal cord (‘sc’). Whole-mount embryos treated with RNase A served as negative control for fluorescence. (F) FISH using LNA probes for Ginir (Green) or Giniras (Red) on embryo sections of 13.5 and 14.5 dpc embryos showing differential expression in forebrain (‘fb’), midbrain (‘mb’), hypothalamus (‘ht’), and limbs (‘li’). Embryo sections treated with RNase A served as a negative control. dpc, days post coitum; EST, expressed sequence tag; FAM, fluorescein amidite; FISH, fluorescence in situ hybridisation; Gapdh, glyceride 3-phosphate dehydrogenase; Ginir, Genomic Instability Inducing RNA; Giniras, antisense RNA of Ginir; ISH, in situ hybridisation; LNA, locked nucleic acid; PCR, polymerase chain reaction; RPA, ribonuclease protection assay; RT-PCR, reverse transcription PCR.

Techniques Used: Expressing, Sequencing, Recombinase Polymerase Amplification, Isolation, Polymerase Chain Reaction, Generated, Activity Assay, Mouse Assay, Reverse Transcription Polymerase Chain Reaction, In Situ Hybridization, Negative Control, Fluorescence, Fluorescence In Situ Hybridization, In Situ, Hybridization

33) Product Images from "Noncoding RNA Ginir functions as an oncogene by associating with centrosomal proteins"

Article Title: Noncoding RNA Ginir functions as an oncogene by associating with centrosomal proteins

Journal: PLoS Biology

doi: 10.1371/journal.pbio.2004204

Ginir RNA impairs interaction between Cep112 and Brca1 proteins leading to genomic instability. (A) RNA pull-down with biotinylated Ginir RNA in NIH/3T3 cells followed by western blotting with Brca1 antibody (sc-646, Santa Cruz). Pull-down with unbiotinylated RNA probe served as control for nonspecific binding. (B-D) RIP performed using both Cep112 and Brca1 antibodies followed by RNA isolation and RT-PCR with Ginir specific primers (G2F-G2R) in NIH/3T3-GinirA (B) and NIH/3T3-GinirB (C) cells. RIP assay was also followed by RT-PCR using nonspecific primers for U6 snRNA (D). Anti-IgG IP served as control for nonspecific interaction. (E) In silico model of Cep112 and Brca1 interaction generated through computational docking using ZDOCK tool. (F and G) Co-IP of Cep112 and Brca1 proteins in NIH/3T3-EV cells. IP was performed with Brca1 antibody (sc-646, Santa Cruz) followed by immunoblotting with Cep112 antibody (sc-246162, Santa Cruz) (F) and vice versa (G). Anti-IgG IP served as a control for nonspecific binding to the antibody. (H and I) Western blotting with Cep112 antibody (24928-1-AP, Proteintech) for validation of Flag-Cep112 overexpression in NIH/3T3 cells (H). Fifty μg of whole-cell protein lysates from each of the mentioned cell lines was loaded on 7% SDS-PAGE. Tubulin served as internal loading control (I). (J) Co-IP of Brca1 and Flag-Cep112 in NIH-Flag-Cep112 cells. IP was performed with Flag1 antibody followed by immunoblotting with Brca1 antibody (20649-1-AP, Proteintech). Anti-IgG IP served as a control for nonspecific binding to the antibody. (K) Co-IP of Brca1 and Cep112 proteins in NIH/3T3-EV, NIH/3T3-Ginir, and NIH-Ginir-shGinir2 cells. IP was performed with Brca1 antibody (sc-646, Santa Cruz) followed by immunoblotting with Cep112 antibody (24928-1-AP, Proteintech). Anti-IgG IP served as a control for nonspecific binding to the antibody. (L) Confocal images showing colocalisation of Brca1 with γ-tubulin in NIH/3T3-EV cell line. Nuclei were stained with DAPI. Scale bars, 10 μm. (M) Confocal images showing colocalisation of Brca1 protein with Cep112 protein in NIH/3T3-EV cell line. Nuclei were stained with DAPI. Scale bars, 20 μm. (N) Confocal imaging for Brca1 expression in NIH/3T3-EV and NIH/3T3-Ginir cells. Scale bars, 20 μm. (O) RNA-FISH using Ginir-specific probe (probe 1, FAM labelled) in NIH/3T3-EV cells visualised by confocal imaging. Scale bars, 20 μm. (P) Co-IP of Brca1 and Cep112 in NIH/3T3-Ginir(C) cells wherein lysates were treated independently with RNase (A, H, and III mix) or RNasin. Both RNase- and RNasin-treated lysates were immunoprecipitated with Brca1 antibody (sc-646, Santa Cruz) and blotted using Cep112 antibody (sc-246163, Santa Cruz). IP with anti-IgG served as control. Brca1, breast cancer type 1 susceptibility protein; Cep112, centrosomal protein 112; FAM, fluorescein amidite; Ginir, Genomic Instability Inducing RNA; IgG, immunoglobulin G; IP, immunoprecipitation; RIP, RNA-immunoprecipitation; RNasin, RNase inhibitor; RT-PCR, reverse transcription polymerase chain reaction; snRNA, small nuclear RNA.
Figure Legend Snippet: Ginir RNA impairs interaction between Cep112 and Brca1 proteins leading to genomic instability. (A) RNA pull-down with biotinylated Ginir RNA in NIH/3T3 cells followed by western blotting with Brca1 antibody (sc-646, Santa Cruz). Pull-down with unbiotinylated RNA probe served as control for nonspecific binding. (B-D) RIP performed using both Cep112 and Brca1 antibodies followed by RNA isolation and RT-PCR with Ginir specific primers (G2F-G2R) in NIH/3T3-GinirA (B) and NIH/3T3-GinirB (C) cells. RIP assay was also followed by RT-PCR using nonspecific primers for U6 snRNA (D). Anti-IgG IP served as control for nonspecific interaction. (E) In silico model of Cep112 and Brca1 interaction generated through computational docking using ZDOCK tool. (F and G) Co-IP of Cep112 and Brca1 proteins in NIH/3T3-EV cells. IP was performed with Brca1 antibody (sc-646, Santa Cruz) followed by immunoblotting with Cep112 antibody (sc-246162, Santa Cruz) (F) and vice versa (G). Anti-IgG IP served as a control for nonspecific binding to the antibody. (H and I) Western blotting with Cep112 antibody (24928-1-AP, Proteintech) for validation of Flag-Cep112 overexpression in NIH/3T3 cells (H). Fifty μg of whole-cell protein lysates from each of the mentioned cell lines was loaded on 7% SDS-PAGE. Tubulin served as internal loading control (I). (J) Co-IP of Brca1 and Flag-Cep112 in NIH-Flag-Cep112 cells. IP was performed with Flag1 antibody followed by immunoblotting with Brca1 antibody (20649-1-AP, Proteintech). Anti-IgG IP served as a control for nonspecific binding to the antibody. (K) Co-IP of Brca1 and Cep112 proteins in NIH/3T3-EV, NIH/3T3-Ginir, and NIH-Ginir-shGinir2 cells. IP was performed with Brca1 antibody (sc-646, Santa Cruz) followed by immunoblotting with Cep112 antibody (24928-1-AP, Proteintech). Anti-IgG IP served as a control for nonspecific binding to the antibody. (L) Confocal images showing colocalisation of Brca1 with γ-tubulin in NIH/3T3-EV cell line. Nuclei were stained with DAPI. Scale bars, 10 μm. (M) Confocal images showing colocalisation of Brca1 protein with Cep112 protein in NIH/3T3-EV cell line. Nuclei were stained with DAPI. Scale bars, 20 μm. (N) Confocal imaging for Brca1 expression in NIH/3T3-EV and NIH/3T3-Ginir cells. Scale bars, 20 μm. (O) RNA-FISH using Ginir-specific probe (probe 1, FAM labelled) in NIH/3T3-EV cells visualised by confocal imaging. Scale bars, 20 μm. (P) Co-IP of Brca1 and Cep112 in NIH/3T3-Ginir(C) cells wherein lysates were treated independently with RNase (A, H, and III mix) or RNasin. Both RNase- and RNasin-treated lysates were immunoprecipitated with Brca1 antibody (sc-646, Santa Cruz) and blotted using Cep112 antibody (sc-246163, Santa Cruz). IP with anti-IgG served as control. Brca1, breast cancer type 1 susceptibility protein; Cep112, centrosomal protein 112; FAM, fluorescein amidite; Ginir, Genomic Instability Inducing RNA; IgG, immunoglobulin G; IP, immunoprecipitation; RIP, RNA-immunoprecipitation; RNasin, RNase inhibitor; RT-PCR, reverse transcription polymerase chain reaction; snRNA, small nuclear RNA.

Techniques Used: Western Blot, Binding Assay, Isolation, Reverse Transcription Polymerase Chain Reaction, In Silico, Generated, Co-Immunoprecipitation Assay, Over Expression, SDS Page, Staining, Imaging, Expressing, Fluorescence In Situ Hybridization, Immunoprecipitation

Expression of Ginir/Giniras transcripts during mouse embryonic development and in adult tissues. ). (B) RPA of RNA isolated from NIH/3T3 cells using PCR-generated sense or antisense riboprobes specific to Ginir sequence. Yeast total RNA served as control for RNase A/T1 activity. (C and D) Expression of Ginir/Giniras RNA in different stages of development (dpc) of mouse embryos (C) and in tissues from different organs of adult mice (D) using strand-specific cDNA synthesis and RT-PCR with G1F-G1R primers. Gapdh served as internal loading control. (E) Whole-mount ISH using LNA probes for Ginir (FAM labelled, green) or Giniras (TexRed labelled, red) on 10.5 dpc mouse embryos showing differential expression in brain (‘br’), forelimbs (‘fl’), and spinal cord (‘sc’). Whole-mount embryos treated with RNase A served as negative control for fluorescence. (F) FISH using LNA probes for Ginir (Green) or Giniras (Red) on embryo sections of 13.5 and 14.5 dpc embryos showing differential expression in forebrain (‘fb’), midbrain (‘mb’), hypothalamus (‘ht’), and limbs (‘li’). Embryo sections treated with RNase A served as a negative control. dpc, days post coitum; EST, expressed sequence tag; FAM, fluorescein amidite; FISH, fluorescence in situ hybridisation; Gapdh, glyceride 3-phosphate dehydrogenase; Ginir, Genomic Instability Inducing RNA; Giniras, antisense RNA of Ginir; ISH, in situ hybridisation; LNA, locked nucleic acid; PCR, polymerase chain reaction; RPA, ribonuclease protection assay; RT-PCR, reverse transcription PCR.
Figure Legend Snippet: Expression of Ginir/Giniras transcripts during mouse embryonic development and in adult tissues. ). (B) RPA of RNA isolated from NIH/3T3 cells using PCR-generated sense or antisense riboprobes specific to Ginir sequence. Yeast total RNA served as control for RNase A/T1 activity. (C and D) Expression of Ginir/Giniras RNA in different stages of development (dpc) of mouse embryos (C) and in tissues from different organs of adult mice (D) using strand-specific cDNA synthesis and RT-PCR with G1F-G1R primers. Gapdh served as internal loading control. (E) Whole-mount ISH using LNA probes for Ginir (FAM labelled, green) or Giniras (TexRed labelled, red) on 10.5 dpc mouse embryos showing differential expression in brain (‘br’), forelimbs (‘fl’), and spinal cord (‘sc’). Whole-mount embryos treated with RNase A served as negative control for fluorescence. (F) FISH using LNA probes for Ginir (Green) or Giniras (Red) on embryo sections of 13.5 and 14.5 dpc embryos showing differential expression in forebrain (‘fb’), midbrain (‘mb’), hypothalamus (‘ht’), and limbs (‘li’). Embryo sections treated with RNase A served as a negative control. dpc, days post coitum; EST, expressed sequence tag; FAM, fluorescein amidite; FISH, fluorescence in situ hybridisation; Gapdh, glyceride 3-phosphate dehydrogenase; Ginir, Genomic Instability Inducing RNA; Giniras, antisense RNA of Ginir; ISH, in situ hybridisation; LNA, locked nucleic acid; PCR, polymerase chain reaction; RPA, ribonuclease protection assay; RT-PCR, reverse transcription PCR.

Techniques Used: Expressing, Recombinase Polymerase Amplification, Isolation, Polymerase Chain Reaction, Generated, Sequencing, Activity Assay, Mouse Assay, Reverse Transcription Polymerase Chain Reaction, In Situ Hybridization, Negative Control, Fluorescence, Fluorescence In Situ Hybridization, In Situ, Hybridization

34) Product Images from "RNA-dependent stabilization of SUV39H1 at constitutive heterochromatin"

Article Title: RNA-dependent stabilization of SUV39H1 at constitutive heterochromatin

Journal: eLife

doi: 10.7554/eLife.25299

HP1α localization correlates with SUV39H1 localization, despite no change in H3K9me3 levels. ( A ) Representative images of HP1α and SUV39H1-GFP localization on HeLa mitotic chromosomes after RNase treatment. Expression of SUV39H1-GFP was induced or not for 6 hr (+/- dox), then mitotic cells were spun onto coverslips and incubated without RNase, with RNase A, or with RNase A plus RNase inhibitors. Cells were then stained for DNA (blue), with an anti-GFP antibody to detect SUV39H1-GFP, HP1α (green), and HEC1 to mark centromeres (red). ( B ) Quantification of HP1α at pericentric regions from experiment shown in A. Each experiment was normalized to the untreated -dox measurement. Bars are the average of n = 5 separate experiments, 15 cells quantified per condition per experiment, error bars represent standard error. ( C ) Quantification of H3K9me3 at pericentric regions in HeLa cells with or without exogenous expression of SUV39H1-GFP for 6 hr (+/- dox). Bars are the average of n = 3 separate experiments, 15 cells quantified per condition per experiment, error bars represent standard error. ( D ) HP1α localization is reduced in cells expressing mutant SUV39H1-GFP compared to cells expressing WT SUV39H1-GFP. Expression of SUV39H1-GFP, WT or mutants, was induced or not for 6 hr (-/+ dox) in HeLa Flp in TREx cell lines. Mitotic cells were spun onto coverslips and stained for DNA (blue), HP1α (green), and HEC1 to mark centromeres (red). ( E ) Quantification of HP1α at pericentric regions from experiment shown in D. Each experiment was normalized to the wildtype measurement. Bars are the average of n = 5 separate experiments, 15 cells quantified per condition per experiment, error bars represent standard error. P values were calculated with paired, two-tailed t tests. DOI: http://dx.doi.org/10.7554/eLife.25299.015
Figure Legend Snippet: HP1α localization correlates with SUV39H1 localization, despite no change in H3K9me3 levels. ( A ) Representative images of HP1α and SUV39H1-GFP localization on HeLa mitotic chromosomes after RNase treatment. Expression of SUV39H1-GFP was induced or not for 6 hr (+/- dox), then mitotic cells were spun onto coverslips and incubated without RNase, with RNase A, or with RNase A plus RNase inhibitors. Cells were then stained for DNA (blue), with an anti-GFP antibody to detect SUV39H1-GFP, HP1α (green), and HEC1 to mark centromeres (red). ( B ) Quantification of HP1α at pericentric regions from experiment shown in A. Each experiment was normalized to the untreated -dox measurement. Bars are the average of n = 5 separate experiments, 15 cells quantified per condition per experiment, error bars represent standard error. ( C ) Quantification of H3K9me3 at pericentric regions in HeLa cells with or without exogenous expression of SUV39H1-GFP for 6 hr (+/- dox). Bars are the average of n = 3 separate experiments, 15 cells quantified per condition per experiment, error bars represent standard error. ( D ) HP1α localization is reduced in cells expressing mutant SUV39H1-GFP compared to cells expressing WT SUV39H1-GFP. Expression of SUV39H1-GFP, WT or mutants, was induced or not for 6 hr (-/+ dox) in HeLa Flp in TREx cell lines. Mitotic cells were spun onto coverslips and stained for DNA (blue), HP1α (green), and HEC1 to mark centromeres (red). ( E ) Quantification of HP1α at pericentric regions from experiment shown in D. Each experiment was normalized to the wildtype measurement. Bars are the average of n = 5 separate experiments, 15 cells quantified per condition per experiment, error bars represent standard error. P values were calculated with paired, two-tailed t tests. DOI: http://dx.doi.org/10.7554/eLife.25299.015

Techniques Used: Expressing, Incubation, Staining, Mutagenesis, Two Tailed Test

Characterization of chromosome-associated pericentric RNA. ( A ) RNase sensitivity of pericentric RNA. EU-labeled mitotic HeLa cells were spun onto coverslips and incubated with or without RNase A, RNase H, or RNase III as indicated, then stained for DNA (blue), EU-RNA (green) and CENP-A (red) to mark centromeres. ( B, C ) Representative images of different types of human cell lines, showing RNA localization on mitotic chromosomes. Cells were labeled with EU for 12 hr, then mitotic cells were spun onto coverslips and stained for DNA (blue), EU-RNA (green), and H3K9me3 (red). ( B ) shows cell lines tested that, like HeLa cells, show RNA concentrated around centromeres. ( C ) shows cell lines tested that show no apparent concentration of RNA around centromeres. ( D ) EU-RNA staining on DLD-1 chromosomes. Cells were labeled with EU and stained as described above. Although α-satellite RNA is detected on DLD-1 chromosomes by RNA FISH, there is little detectable EU-RNA signal. ( E ) β-satellite and Satellite III DNA and RNA on human mitotic chromosomes. Mitotic DLD-1 cells were spread onto coverslips, RNA or DNA FISH was performed to detect Satellite III (green) and β-satellite (red) sequences, and then chromosomes were stained for DNA (blue). The β-satellite probe recognizes sequences on chromosomes 13, 14, 15, 21, and 22, and the Satellite III probe recognizes sequences on chromosomes 14 and 22. ( F ) RNA FISH for D1Z5, a chromosome 1 specific α-satellite array, on HeLa mitotic chromosomes. Mitotic HeLa cells were spread onto coverslips, RNA FISH was performed to detect chromosome 1-specific D1Z5 α-satellite sequences (green), and chromosomes were stained for HEC1 to mark centromeres (red) and with Hoechst to stain for DNA (blue). ( G ) RNA FISH on DLD-1 chromosomes with a probe specific to an α-satellite array on chromosomes 13 and 21. Mitotic DLD-1 cells were spread onto coverslips, RNA FISH was performed to detect α-satellite 13/21 (green), and then chromosomes were stained for DNA (blue) and H3K9me3 (red). Line scans of all four 13 and 21 chromosomes show localization of α-satellite RNA and H3K9me3. White arrows delineate direction of line scan, and the labeling of homologous chromosomes as ‘homolog 1’ or ‘homolog 2’ is arbitrary. The Y-axis represents the pixel intensity along the drawn line. DOI: http://dx.doi.org/10.7554/eLife.25299.004
Figure Legend Snippet: Characterization of chromosome-associated pericentric RNA. ( A ) RNase sensitivity of pericentric RNA. EU-labeled mitotic HeLa cells were spun onto coverslips and incubated with or without RNase A, RNase H, or RNase III as indicated, then stained for DNA (blue), EU-RNA (green) and CENP-A (red) to mark centromeres. ( B, C ) Representative images of different types of human cell lines, showing RNA localization on mitotic chromosomes. Cells were labeled with EU for 12 hr, then mitotic cells were spun onto coverslips and stained for DNA (blue), EU-RNA (green), and H3K9me3 (red). ( B ) shows cell lines tested that, like HeLa cells, show RNA concentrated around centromeres. ( C ) shows cell lines tested that show no apparent concentration of RNA around centromeres. ( D ) EU-RNA staining on DLD-1 chromosomes. Cells were labeled with EU and stained as described above. Although α-satellite RNA is detected on DLD-1 chromosomes by RNA FISH, there is little detectable EU-RNA signal. ( E ) β-satellite and Satellite III DNA and RNA on human mitotic chromosomes. Mitotic DLD-1 cells were spread onto coverslips, RNA or DNA FISH was performed to detect Satellite III (green) and β-satellite (red) sequences, and then chromosomes were stained for DNA (blue). The β-satellite probe recognizes sequences on chromosomes 13, 14, 15, 21, and 22, and the Satellite III probe recognizes sequences on chromosomes 14 and 22. ( F ) RNA FISH for D1Z5, a chromosome 1 specific α-satellite array, on HeLa mitotic chromosomes. Mitotic HeLa cells were spread onto coverslips, RNA FISH was performed to detect chromosome 1-specific D1Z5 α-satellite sequences (green), and chromosomes were stained for HEC1 to mark centromeres (red) and with Hoechst to stain for DNA (blue). ( G ) RNA FISH on DLD-1 chromosomes with a probe specific to an α-satellite array on chromosomes 13 and 21. Mitotic DLD-1 cells were spread onto coverslips, RNA FISH was performed to detect α-satellite 13/21 (green), and then chromosomes were stained for DNA (blue) and H3K9me3 (red). Line scans of all four 13 and 21 chromosomes show localization of α-satellite RNA and H3K9me3. White arrows delineate direction of line scan, and the labeling of homologous chromosomes as ‘homolog 1’ or ‘homolog 2’ is arbitrary. The Y-axis represents the pixel intensity along the drawn line. DOI: http://dx.doi.org/10.7554/eLife.25299.004

Techniques Used: Labeling, Incubation, Staining, Concentration Assay, Fluorescence In Situ Hybridization

Characterization of SUV39 DKO human cells. ( A ) Western blot analysis of HeLa SUV39 DKO cells, assessing total SUV39H1, HP1α, tubulin, and H3K9me3 levels. ( B ) Analysis of SUV39H2 loci in HeLa SUV39 DKO cells. A section of the SUV39H2 gene surrounding the targeted Cas9 cut site was PCRed from genomic DNA and sequenced. Two mutant alleles were identified, and both lead to an early translation termination of SUV39H2. ( C ) α-satellite RNA levels in HeLa SUV39 DKO cells were measured by reverse transcription followed by quantitative PCR (RT-qPCR). Total RNA was isolated from HeLa control and HeLa SUV39 DKO cells, then reverse transcribed and amplified with α-satellite or GAPDH primers. Shown is average of 5 independent measurements, normalized to control cells, error bars are standard error. ( D ) H3K9me3 localization in control or SUV39 DKO HeLa cells. Mitotic cells were spread onto coverslips, then stained for DNA (blue), H3K9me3 (green), and HEC1 (red) to mark core centromere/kinetochore regions. ( E ) HP1α localization in control or SUV39 DKO HeLa cells. Mitotic cells were spread onto coverslips, then stained for DNA (blue), HP1α (green), and HEC1 (red) to mark core centromere/kinetochore regions. ( F ) Analysis of repetitive RNAs in HeLa SUV39 DKO cells. Total RNA was isolated from HeLa control and HeLa SUV39 DKO cells, and a cDNA library was generated and sequenced. Fold change in RNAs (SUV39 DKO / control) transcribed from different repeat types were plotted as a rank order from highest to lowest. Repeat types with over 300 reads were included. The horizontal gray dotted line represents a cutoff of 2 standard deviations from the mean of the dataset. Brown dots represent repeat types that fall under two standard deviations from the mean. Repeats that were enriched more than two standard deviations from the mean are labeled, and colors represent the RepeatMasker broad repeat class to which that repeat type belongs. ( G ) Comparative analysis of non-repetitive RNA levels in HeLa control and HeLa SUV39 DKO cells. RNA was isolated and sequenced as described in F , and non-repetitive (uniquely mapping) RNAs were analyzed. FPKM: Fragments Per Kilobase of transcript per Million mapped reads. ( H ) Western blot analysis of DLD-1 SUV39 DKO cells, assessing total SUV39H1, HP1α, tubulin, and H3K9me3 levels. ( I ) Analysis of SUV39H2 loci in DLD-1 SUV39 DKO cells. A section of the SUV39H2 gene surrounding the targeted Cas9 cut site was PCRed from genomic DNA and subjected to MiSeq sequencing. Two mutant alleles were identified, and both lead to an early stop in the translation of SUV39H2. ( J ) α-satellite RNA levels in DLD-1 SUV39 DKO cells were measured by reverse transcription followed by quantitative PCR (RT-qPCR). Total RNA was isolated from DLD-1 control and DLD-1 SUV39 DKO cells, then reverse transcribed and amplified with α-satellite or GAPDH primers. Shown is average of 5 independent measurements, normalized to control cells, error bars are standard error. ( K ) H3K9me3 localization in control or SUV39 DKO DLD-1 cells. Mitotic cells were spread onto coverslips, then stained for DNA (blue), H3K9me3 (green), and HEC1 (red) to mark core centromere/kinetochore regions. ( L ) α-satellite RNA localization in control or SUV39 DKO DLD-1 cells. Mitotic cells were spread onto coverslips, then stained for DNA (blue), α-satellite (D1Z5 probe, green), and HEC1 (red) to mark core centromere/kinetochore regions. ( M ) Western blot showing SUV39H1 and histone H3 protein levels in cell lysates treated with RNase A or RNase A plus RNase inhibitors. DOI: http://dx.doi.org/10.7554/eLife.25299.007
Figure Legend Snippet: Characterization of SUV39 DKO human cells. ( A ) Western blot analysis of HeLa SUV39 DKO cells, assessing total SUV39H1, HP1α, tubulin, and H3K9me3 levels. ( B ) Analysis of SUV39H2 loci in HeLa SUV39 DKO cells. A section of the SUV39H2 gene surrounding the targeted Cas9 cut site was PCRed from genomic DNA and sequenced. Two mutant alleles were identified, and both lead to an early translation termination of SUV39H2. ( C ) α-satellite RNA levels in HeLa SUV39 DKO cells were measured by reverse transcription followed by quantitative PCR (RT-qPCR). Total RNA was isolated from HeLa control and HeLa SUV39 DKO cells, then reverse transcribed and amplified with α-satellite or GAPDH primers. Shown is average of 5 independent measurements, normalized to control cells, error bars are standard error. ( D ) H3K9me3 localization in control or SUV39 DKO HeLa cells. Mitotic cells were spread onto coverslips, then stained for DNA (blue), H3K9me3 (green), and HEC1 (red) to mark core centromere/kinetochore regions. ( E ) HP1α localization in control or SUV39 DKO HeLa cells. Mitotic cells were spread onto coverslips, then stained for DNA (blue), HP1α (green), and HEC1 (red) to mark core centromere/kinetochore regions. ( F ) Analysis of repetitive RNAs in HeLa SUV39 DKO cells. Total RNA was isolated from HeLa control and HeLa SUV39 DKO cells, and a cDNA library was generated and sequenced. Fold change in RNAs (SUV39 DKO / control) transcribed from different repeat types were plotted as a rank order from highest to lowest. Repeat types with over 300 reads were included. The horizontal gray dotted line represents a cutoff of 2 standard deviations from the mean of the dataset. Brown dots represent repeat types that fall under two standard deviations from the mean. Repeats that were enriched more than two standard deviations from the mean are labeled, and colors represent the RepeatMasker broad repeat class to which that repeat type belongs. ( G ) Comparative analysis of non-repetitive RNA levels in HeLa control and HeLa SUV39 DKO cells. RNA was isolated and sequenced as described in F , and non-repetitive (uniquely mapping) RNAs were analyzed. FPKM: Fragments Per Kilobase of transcript per Million mapped reads. ( H ) Western blot analysis of DLD-1 SUV39 DKO cells, assessing total SUV39H1, HP1α, tubulin, and H3K9me3 levels. ( I ) Analysis of SUV39H2 loci in DLD-1 SUV39 DKO cells. A section of the SUV39H2 gene surrounding the targeted Cas9 cut site was PCRed from genomic DNA and subjected to MiSeq sequencing. Two mutant alleles were identified, and both lead to an early stop in the translation of SUV39H2. ( J ) α-satellite RNA levels in DLD-1 SUV39 DKO cells were measured by reverse transcription followed by quantitative PCR (RT-qPCR). Total RNA was isolated from DLD-1 control and DLD-1 SUV39 DKO cells, then reverse transcribed and amplified with α-satellite or GAPDH primers. Shown is average of 5 independent measurements, normalized to control cells, error bars are standard error. ( K ) H3K9me3 localization in control or SUV39 DKO DLD-1 cells. Mitotic cells were spread onto coverslips, then stained for DNA (blue), H3K9me3 (green), and HEC1 (red) to mark core centromere/kinetochore regions. ( L ) α-satellite RNA localization in control or SUV39 DKO DLD-1 cells. Mitotic cells were spread onto coverslips, then stained for DNA (blue), α-satellite (D1Z5 probe, green), and HEC1 (red) to mark core centromere/kinetochore regions. ( M ) Western blot showing SUV39H1 and histone H3 protein levels in cell lysates treated with RNase A or RNase A plus RNase inhibitors. DOI: http://dx.doi.org/10.7554/eLife.25299.007

Techniques Used: Western Blot, Mutagenesis, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Isolation, Amplification, Staining, cDNA Library Assay, Generated, Labeling, Sequencing

35) Product Images from "Structure of a bacterial homolog of vitamin K epoxide reductase"

Article Title: Structure of a bacterial homolog of vitamin K epoxide reductase

Journal: Nature

doi: 10.1038/nature08720

In vitro reconstitution of vitamin K-dependent oxidative folding a , Purified Synechococcus protein, containing the VKOR and Trx-like domains, was incubated with reduced, denatured RNAse A and vitamin K1 (Vit K1) (lanes 9, 10). The reaction was quenched with AMS, which modifies free sulfhydryl groups. Samples were analyzed by SDS-PAGE and Coomassie staining. Controls were performed without Vit K1 (in ethanol, EtOH) or VKOR, as indicated. RNAse A was also oxidized with glutathione (GSH/GSSG) (lanes 3,4). b , As in a , but with VKOR serially diluted. c , As in a , but with wild type (WT) or mutants in cysteines in the predicted electron transfer pathway ( Fig. 4 ). d , As in c , except reduction of Vit K1 was followed in a fluorometer, using reduced RNAse or dithiothreitol (DTT) as reductant. Error bars represent standard error of mean instrumental fluorescence variation.
Figure Legend Snippet: In vitro reconstitution of vitamin K-dependent oxidative folding a , Purified Synechococcus protein, containing the VKOR and Trx-like domains, was incubated with reduced, denatured RNAse A and vitamin K1 (Vit K1) (lanes 9, 10). The reaction was quenched with AMS, which modifies free sulfhydryl groups. Samples were analyzed by SDS-PAGE and Coomassie staining. Controls were performed without Vit K1 (in ethanol, EtOH) or VKOR, as indicated. RNAse A was also oxidized with glutathione (GSH/GSSG) (lanes 3,4). b , As in a , but with VKOR serially diluted. c , As in a , but with wild type (WT) or mutants in cysteines in the predicted electron transfer pathway ( Fig. 4 ). d , As in c , except reduction of Vit K1 was followed in a fluorometer, using reduced RNAse or dithiothreitol (DTT) as reductant. Error bars represent standard error of mean instrumental fluorescence variation.

Techniques Used: In Vitro, Purification, Incubation, Affinity Magnetic Separation, SDS Page, Staining, Fluorescence

36) Product Images from "Evidence that Lin28 stimulates translation by recruiting RNA helicase A to polysomes"

Article Title: Evidence that Lin28 stimulates translation by recruiting RNA helicase A to polysomes

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkq1350

The N- and C-terminal regions of RHA interact with Lin28. ( A ) Domain organization of human RHA protein. Double-stranded RNA binding domain I and II (dsRBD I and II), C-terminal domain rich in arginine-glycine-glycine (RGG) repeats and the Walker helicase motifs of the conserved DEAD-box RNA helicases are depicted. Numbers indicate corresponding amino acid residue. ( B ) GST pulldown results. HEK293 cell lysate containing Flag-Lin28 was incubated with bacterial lysate containing the indicated recombinant RHA domains or GST alone in the presence of RNase A, followed by GST pulldown assays. Left panel, anti-Flag and anti-PABP antibodies were used in the upper and lower blots, respectively. Input was 0.5% of the total amount of proteins used for each GST pulldown. Right panel, Coomassie staining determined comparable amounts of the recombinant proteins used in the GST pulldown assays. 1% of the input was loaded in each lane. Molecular size markers are on the right. ( C ) Flag-Lin28 and Flag-N300 were co-transfected into HEK293 cells. Co-IP was carried out in the presence of RNase A 24 h later using anti-Lin28 antibody to bring down Flag-Lin28 together with its associated proteins, followed by western blot analysis. Antibodies used in the western blot were anti-RHA (top two blots, note, this antibody recognizes both full-length RHA and Flag-N300), anti-NXF1 (third blot from top), and anti-Flag M2 (bottom blot). Total proteins (2%) used for each immunoprecipitation was loaded as input.
Figure Legend Snippet: The N- and C-terminal regions of RHA interact with Lin28. ( A ) Domain organization of human RHA protein. Double-stranded RNA binding domain I and II (dsRBD I and II), C-terminal domain rich in arginine-glycine-glycine (RGG) repeats and the Walker helicase motifs of the conserved DEAD-box RNA helicases are depicted. Numbers indicate corresponding amino acid residue. ( B ) GST pulldown results. HEK293 cell lysate containing Flag-Lin28 was incubated with bacterial lysate containing the indicated recombinant RHA domains or GST alone in the presence of RNase A, followed by GST pulldown assays. Left panel, anti-Flag and anti-PABP antibodies were used in the upper and lower blots, respectively. Input was 0.5% of the total amount of proteins used for each GST pulldown. Right panel, Coomassie staining determined comparable amounts of the recombinant proteins used in the GST pulldown assays. 1% of the input was loaded in each lane. Molecular size markers are on the right. ( C ) Flag-Lin28 and Flag-N300 were co-transfected into HEK293 cells. Co-IP was carried out in the presence of RNase A 24 h later using anti-Lin28 antibody to bring down Flag-Lin28 together with its associated proteins, followed by western blot analysis. Antibodies used in the western blot were anti-RHA (top two blots, note, this antibody recognizes both full-length RHA and Flag-N300), anti-NXF1 (third blot from top), and anti-Flag M2 (bottom blot). Total proteins (2%) used for each immunoprecipitation was loaded as input.

Techniques Used: RNA Binding Assay, Incubation, Recombinant, Staining, Transfection, Co-Immunoprecipitation Assay, Western Blot, Immunoprecipitation

C-terminus deletion reduces Lin28′s ability to interact with RHA. ( A ) Schematic of wild-type and mutant Lin28 protein. Numbers are in amino acids. ( B ) Flag-Lin28, Flag-Lin28ΔN, Flag-Lin28ΔC or empty vector were each transfected into HEK293 cells. Co-IP was carried out in the presence of RNase A using anti-Flag antibody. Resulting protein complexes were resolved by SDS–PAGE, followed by western blot analysis. Anti-RHA and polyclonal anti-Flag antibody were used in the top and bottom blots, respectively. Three percent of input was loaded. ( C ) Flag-Lin28ΔC was transfected into HEK293 cells. Cell lysate containing Flag-Lin28ΔC was incubated with bacterial lysate containing the indicated recombinant RHA domains or GST, followed by GST pulldown assays. Top panel, anti-Flag M2 antibody was used to detect Flag-Lin28ΔC. About 0.5% of the input was loaded in lane 1. Bottom panel, Coomassie staining of the recombinant proteins used in the GST pulldown assays. About 1% of the input was loaded in each lane. Molecular size markers are on the right.
Figure Legend Snippet: C-terminus deletion reduces Lin28′s ability to interact with RHA. ( A ) Schematic of wild-type and mutant Lin28 protein. Numbers are in amino acids. ( B ) Flag-Lin28, Flag-Lin28ΔN, Flag-Lin28ΔC or empty vector were each transfected into HEK293 cells. Co-IP was carried out in the presence of RNase A using anti-Flag antibody. Resulting protein complexes were resolved by SDS–PAGE, followed by western blot analysis. Anti-RHA and polyclonal anti-Flag antibody were used in the top and bottom blots, respectively. Three percent of input was loaded. ( C ) Flag-Lin28ΔC was transfected into HEK293 cells. Cell lysate containing Flag-Lin28ΔC was incubated with bacterial lysate containing the indicated recombinant RHA domains or GST, followed by GST pulldown assays. Top panel, anti-Flag M2 antibody was used to detect Flag-Lin28ΔC. About 0.5% of the input was loaded in lane 1. Bottom panel, Coomassie staining of the recombinant proteins used in the GST pulldown assays. About 1% of the input was loaded in each lane. Molecular size markers are on the right.

Techniques Used: Mutagenesis, Plasmid Preparation, Transfection, Co-Immunoprecipitation Assay, SDS Page, Western Blot, Incubation, Recombinant, Staining

Lin28 interacts with RHA in PA-1 cells. Lin28-containing protein complexes were immunoprecipitated in the presence of excess amounts of RNase A from PA-1 cells using anti-Lin28 or pre-immune IgG (as a negative control for non-specific binding). Co-IP complexes (lanes 1 and 2) and 3% input (lane 3) were resolved by SDS–PAGE, followed by western blot analysis using anti-RHA (top blot) and anti-NXF1 (bottom blot) antiboddies, respectively.
Figure Legend Snippet: Lin28 interacts with RHA in PA-1 cells. Lin28-containing protein complexes were immunoprecipitated in the presence of excess amounts of RNase A from PA-1 cells using anti-Lin28 or pre-immune IgG (as a negative control for non-specific binding). Co-IP complexes (lanes 1 and 2) and 3% input (lane 3) were resolved by SDS–PAGE, followed by western blot analysis using anti-RHA (top blot) and anti-NXF1 (bottom blot) antiboddies, respectively.

Techniques Used: Immunoprecipitation, Negative Control, Binding Assay, Co-Immunoprecipitation Assay, SDS Page, Western Blot

37) Product Images from "Phosphorylation State-Dependent Interactions of Hepadnavirus Core Protein with Host Factors"

Article Title: Phosphorylation State-Dependent Interactions of Hepadnavirus Core Protein with Host Factors

Journal: PLoS ONE

doi: 10.1371/journal.pone.0029566

GST pulldown with  E. coli -derived DCC fusion proteins. GST or GST-DCC fusion proteins were purified from  E. coli . The purified proteins were added to RNase A-treated HEK293T cell lysates. HEK293T cellular proteins that were pulled down by the fusion proteins were visualized by silver staining ( A , lanes 1–4) and western blotting ( B , lanes 1–4). For comparison, the same GST-DCC fusion proteins were expressed and purified from HEK293T cells and the co-purifying cellular proteins were shown in  A    B , lanes 5–7. *, non-specific background bands that appeared in all samples, including GST alone.
Figure Legend Snippet: GST pulldown with E. coli -derived DCC fusion proteins. GST or GST-DCC fusion proteins were purified from E. coli . The purified proteins were added to RNase A-treated HEK293T cell lysates. HEK293T cellular proteins that were pulled down by the fusion proteins were visualized by silver staining ( A , lanes 1–4) and western blotting ( B , lanes 1–4). For comparison, the same GST-DCC fusion proteins were expressed and purified from HEK293T cells and the co-purifying cellular proteins were shown in A B , lanes 5–7. *, non-specific background bands that appeared in all samples, including GST alone.

Techniques Used: Derivative Assay, Droplet Countercurrent Chromatography, Purification, Silver Staining, Western Blot

Metabolic labeling of GST-DCC constructs in HEK293T cells. Cells were transfected with the indicated plasmids expressing either GST or GST-DCC fusion proteins, then were metabolically labeled with either [ 35 S]cysteine/methionine or [ 32 P]orthophosphate on the third day post-transfection. A. 35 S- or B. 32 P-labeled proteins were purified without RNase A digestion using GSH affinity resin and were visualized by silver staining ( top panels ) and autoradiography ( bottom panels ). Arrowheads indicate GST or GST-DCC fusion proteins. *, non-specific background bands that appeared in all transfections or untransfected cells.
Figure Legend Snippet: Metabolic labeling of GST-DCC constructs in HEK293T cells. Cells were transfected with the indicated plasmids expressing either GST or GST-DCC fusion proteins, then were metabolically labeled with either [ 35 S]cysteine/methionine or [ 32 P]orthophosphate on the third day post-transfection. A. 35 S- or B. 32 P-labeled proteins were purified without RNase A digestion using GSH affinity resin and were visualized by silver staining ( top panels ) and autoradiography ( bottom panels ). Arrowheads indicate GST or GST-DCC fusion proteins. *, non-specific background bands that appeared in all transfections or untransfected cells.

Techniques Used: Labeling, Droplet Countercurrent Chromatography, Construct, Transfection, Expressing, Metabolic Labelling, Purification, Silver Staining, Autoradiography

GST co-purification and MS analysis. HEK293T cells were transfected with the indicated GST-DCC fusion constructs. GST fusion proteins (short arrows) were purified from RNase A-treated lysates using GSH affinity resin. Co-purifying proteins were resolved by SDS-PAGE and detected by CB staining. Protein bands of interest were excised, subjected to in-gel trypsinization, and identified by mass spectrometry (long arrows). *, non-specific background bands that appeared in all samples, including GST alone.
Figure Legend Snippet: GST co-purification and MS analysis. HEK293T cells were transfected with the indicated GST-DCC fusion constructs. GST fusion proteins (short arrows) were purified from RNase A-treated lysates using GSH affinity resin. Co-purifying proteins were resolved by SDS-PAGE and detected by CB staining. Protein bands of interest were excised, subjected to in-gel trypsinization, and identified by mass spectrometry (long arrows). *, non-specific background bands that appeared in all samples, including GST alone.

Techniques Used: Copurification, Mass Spectrometry, Transfection, Droplet Countercurrent Chromatography, Construct, Purification, SDS Page, Staining

38) Product Images from "Analysis of the oligomeric states of nucleophosmin using size exclusion chromatography"

Article Title: Analysis of the oligomeric states of nucleophosmin using size exclusion chromatography

Journal: Scientific Reports

doi: 10.1038/s41598-018-22359-w

The main component of cellular NPM1 complex is NPM1 and its variant. Both the Triton X-100 soluble and insoluble lysates from HeLa cells were subjected to SEC. Fractions 15 and 16 from the soluble sample (for Group 1), fractions 19 to 21 from the soluble sample (for Group 2), and fractions 15 and 16 from the insoluble sample (for Group 3) were separately pooled. After treatment with or without RNase A, each pooled sample was immunoprecipitated with anti-NPM antibody. Proteins were detected by CBB staining ( Upper panel ) and immunoblot using anti-NPM antibody ( Lower panel ).
Figure Legend Snippet: The main component of cellular NPM1 complex is NPM1 and its variant. Both the Triton X-100 soluble and insoluble lysates from HeLa cells were subjected to SEC. Fractions 15 and 16 from the soluble sample (for Group 1), fractions 19 to 21 from the soluble sample (for Group 2), and fractions 15 and 16 from the insoluble sample (for Group 3) were separately pooled. After treatment with or without RNase A, each pooled sample was immunoprecipitated with anti-NPM antibody. Proteins were detected by CBB staining ( Upper panel ) and immunoblot using anti-NPM antibody ( Lower panel ).

Techniques Used: Variant Assay, Size-exclusion Chromatography, Immunoprecipitation, Staining

Elution profiles of endogenous NPM1 in HeLa cells. ( A ) ( Upper panel ) Elution profiles of Triton X-100 soluble NPM1 and RNA. ( Lower panel ) Elution profiles of Triton X-100 insoluble NPM1 and RNA. Endogenous NPM1 was detected by immunoblot. Protein mass standards are indicated above the  panel : thyroglobulin (669 kDa), ferritin (440 kDa), aldolase (158 kDa), conalbumin (75 kDa), ovalbumin (44 kDa), and Ribonuclease A (13.7 kDa). RNA was detected by ethidium bromide staining. The four NPM1 groups (Group 1 to Group 4) identified by SEC are underlined. ( B ) Schematic representation of sample preparation in C. ( C ) Elution profile of sonication-treated Triton X-100 soluble NPM1 prepared in B.
Figure Legend Snippet: Elution profiles of endogenous NPM1 in HeLa cells. ( A ) ( Upper panel ) Elution profiles of Triton X-100 soluble NPM1 and RNA. ( Lower panel ) Elution profiles of Triton X-100 insoluble NPM1 and RNA. Endogenous NPM1 was detected by immunoblot. Protein mass standards are indicated above the panel : thyroglobulin (669 kDa), ferritin (440 kDa), aldolase (158 kDa), conalbumin (75 kDa), ovalbumin (44 kDa), and Ribonuclease A (13.7 kDa). RNA was detected by ethidium bromide staining. The four NPM1 groups (Group 1 to Group 4) identified by SEC are underlined. ( B ) Schematic representation of sample preparation in C. ( C ) Elution profile of sonication-treated Triton X-100 soluble NPM1 prepared in B.

Techniques Used: Staining, Size-exclusion Chromatography, Sample Prep, Sonication

Purified recombinant NPM1 shows a similar elution profile to that of RNase A-treated cellular NPM1. ( A)  Bacterially expressed His-TEV-NPM1 was purified as described in experimental procedures. Purified proteins showed high purity and did not contain any RNA. ( B)  Elution profile of purified His-TEV-NPM1 proteins.
Figure Legend Snippet: Purified recombinant NPM1 shows a similar elution profile to that of RNase A-treated cellular NPM1. ( A) Bacterially expressed His-TEV-NPM1 was purified as described in experimental procedures. Purified proteins showed high purity and did not contain any RNA. ( B) Elution profile of purified His-TEV-NPM1 proteins.

Techniques Used: Purification, Recombinant

39) Product Images from "Distinct Unfolding and Refolding Pathways of Ribonuclease A Revealed by Heating and Cooling Temperature Jumps"

Article Title: Distinct Unfolding and Refolding Pathways of Ribonuclease A Revealed by Heating and Cooling Temperature Jumps

Journal: Biophysical Journal

doi: 10.1529/biophysj.107.123893

Kinetic transition states of the temperature-induced folding/unfolding reaction of RNase A. Changes of entropy ( A ) and enthalpy ( B ) were determined from the temperature dependence of k f and k u of both heating and cooling T -jumps of wild-type RNase A ( left ) and its Y115W variant ( right ). The direction of the T -jumps is indicated by arrows. Thermodynamic parameters of the folded state were set to zero. U and N denote the unfolded and native states, respectively. # represents the TS ensemble.
Figure Legend Snippet: Kinetic transition states of the temperature-induced folding/unfolding reaction of RNase A. Changes of entropy ( A ) and enthalpy ( B ) were determined from the temperature dependence of k f and k u of both heating and cooling T -jumps of wild-type RNase A ( left ) and its Y115W variant ( right ). The direction of the T -jumps is indicated by arrows. Thermodynamic parameters of the folded state were set to zero. U and N denote the unfolded and native states, respectively. # represents the TS ensemble.

Techniques Used: Variant Assay

Kinetic reaction model for the temperature-triggered folding/unfolding of RNase A. The shaded area indicates the fast kinetic phases observed only for the Y115W variant. Solid and shaded arrows denote heating and cooling  T -jumps, respectively. Undetectable kinetic phases corresponding to conformational unfolding/folding are denoted by the black vertical shape at left.
Figure Legend Snippet: Kinetic reaction model for the temperature-triggered folding/unfolding of RNase A. The shaded area indicates the fast kinetic phases observed only for the Y115W variant. Solid and shaded arrows denote heating and cooling T -jumps, respectively. Undetectable kinetic phases corresponding to conformational unfolding/folding are denoted by the black vertical shape at left.

Techniques Used: Variant Assay

T -jump induced unfolding/folding relaxation kinetics of RNase A Y115W. ( A ) Heating T -jumps started from 45°C. They reached 49, 51, 53, 55, 57, 59, 61, and 63°C. ( B ) Cooling T -jumps started from 63°C. They reached 59, 57, 55, 53, 51, 49, 47, and 45°C. Solution conditions: Y115W at 0.05 mg ml −1 in sodium acetate buffer, 50 mM, pH 5.0.
Figure Legend Snippet: T -jump induced unfolding/folding relaxation kinetics of RNase A Y115W. ( A ) Heating T -jumps started from 45°C. They reached 49, 51, 53, 55, 57, 59, 61, and 63°C. ( B ) Cooling T -jumps started from 63°C. They reached 59, 57, 55, 53, 51, 49, 47, and 45°C. Solution conditions: Y115W at 0.05 mg ml −1 in sodium acetate buffer, 50 mM, pH 5.0.

Techniques Used:

Path-dependent Arrhenius plots. k f ( open symbols ) and k u ( solid symbols ) were determined from heating ( circles ) and cooling ( squares ) T -jumps. The protein was the RNase A variant Y115W. Only the slow kinetic phase is shown.
Figure Legend Snippet: Path-dependent Arrhenius plots. k f ( open symbols ) and k u ( solid symbols ) were determined from heating ( circles ) and cooling ( squares ) T -jumps. The protein was the RNase A variant Y115W. Only the slow kinetic phase is shown.

Techniques Used: Variant Assay

40) Product Images from "Nuclear RNP complex assembly initiates cytoplasmic RNA localization"

Article Title: Nuclear RNP complex assembly initiates cytoplasmic RNA localization

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200309145

Prrp and XStau interact with the localization RNP in the cytoplasm. (A) Stage III/IV oocytes were injected with RNA encoding Prrp-myc, and S10 lysates were prepared from isolated nuclear (lane 1 and lanes 3–5) and cytoplasmic (lane 2 and lanes 6–8) fractions. Immunoprecipitations were performed using control antibody plus S10 lysates (lanes 3 and 6), anti-myc in the absence of lysate (lanes 4 and 7), and anti-myc in the presence of nuclear (lane 5) or cytoplasmic (lane 8) lysates. Total (lanes 1 and 2) and immunoprecipitated (lanes 3–8) Vg1 (top) and VegT (bottom) RNAs were detected by RT-PCR. All samples were run on the same gel, but lane order was changed for presentation in the figure. (B) After expression of Prrp-myc in stage III/IV oocytes, immunoprecipitations were performed using control antibody with Prrp-myc cytoplasmic S10 lysate (lane 2), anti-myc alone (lane 3), and anti-myc plus lysate without (lane 4) or with (lane 5) RNase A treatment. Total (lane 1) and bound proteins (lanes 2–5) were separated by 10% SDS-PAGE and were immunoblotted for Vg1RBP/vera (top), hnRNP I (middle), or XStau (bottom). For each panel all samples were run on the same gel, and adjacent lanes are boxed together. (C) Vg1RBP/vera-FLAG was expressed in stage III/IV oocytes and S10 lysates were prepared from isolated nuclear (lanes 1 and 3–5) and cytoplasmic (lanes 2 and 6–8) fractions. Immunoprecipitations were performed using Sepharose beads plus Vg1RBP/vera S10 lysates (lanes 3 and 6), anti-FLAG beads alone (lanes 4 and 7), and anti-FLAG beads in the presence of nuclear (lane 5) or cytoplasmic (lane 8) lysates. Total (lanes 1 and 2) and bound (lanes 3–8) proteins were separated by 10% SDS-PAGE and immunoblotted for XStau. All samples were run on the same gel, with adjacent lanes boxed together for presentation in the figure.
Figure Legend Snippet: Prrp and XStau interact with the localization RNP in the cytoplasm. (A) Stage III/IV oocytes were injected with RNA encoding Prrp-myc, and S10 lysates were prepared from isolated nuclear (lane 1 and lanes 3–5) and cytoplasmic (lane 2 and lanes 6–8) fractions. Immunoprecipitations were performed using control antibody plus S10 lysates (lanes 3 and 6), anti-myc in the absence of lysate (lanes 4 and 7), and anti-myc in the presence of nuclear (lane 5) or cytoplasmic (lane 8) lysates. Total (lanes 1 and 2) and immunoprecipitated (lanes 3–8) Vg1 (top) and VegT (bottom) RNAs were detected by RT-PCR. All samples were run on the same gel, but lane order was changed for presentation in the figure. (B) After expression of Prrp-myc in stage III/IV oocytes, immunoprecipitations were performed using control antibody with Prrp-myc cytoplasmic S10 lysate (lane 2), anti-myc alone (lane 3), and anti-myc plus lysate without (lane 4) or with (lane 5) RNase A treatment. Total (lane 1) and bound proteins (lanes 2–5) were separated by 10% SDS-PAGE and were immunoblotted for Vg1RBP/vera (top), hnRNP I (middle), or XStau (bottom). For each panel all samples were run on the same gel, and adjacent lanes are boxed together. (C) Vg1RBP/vera-FLAG was expressed in stage III/IV oocytes and S10 lysates were prepared from isolated nuclear (lanes 1 and 3–5) and cytoplasmic (lanes 2 and 6–8) fractions. Immunoprecipitations were performed using Sepharose beads plus Vg1RBP/vera S10 lysates (lanes 3 and 6), anti-FLAG beads alone (lanes 4 and 7), and anti-FLAG beads in the presence of nuclear (lane 5) or cytoplasmic (lane 8) lysates. Total (lanes 1 and 2) and bound (lanes 3–8) proteins were separated by 10% SDS-PAGE and immunoblotted for XStau. All samples were run on the same gel, with adjacent lanes boxed together for presentation in the figure.

Techniques Used: Injection, Isolation, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Expressing, SDS Page

Vg1RBP/vera and hnRNP I interact in vitro and in vivo.  (A) Anti-hnRNP I was used to immunoprecipitate endogenous hnRNP I from oocyte S10 lysate. Bound Vg1RBP/vera was detected by immunoblotting (lane 4). Shown in lane 1 is the total Vg1RBP/vera in the lysate, and immunoprecipitation with protein G beads with or without lysate is shown in lanes 2 and 3, respectively. (B) In vitro–translated Vg1RBP/vera-FLAG was incubated with oocyte S10 lysate. Immunoprecipitations were performed using anti-FLAG beads with S10 lysate (lane 2), anti-FLAG beads with in vitro–translated Vg1RBP/vera-FLAG (lane 3), Sepharose beads with S10 lysate plus in vitro–translated Vg1RBP/vera-FLAG (lane 4), anti-FLAG beads alone (lane 5), and anti-FLAG beads with S10 lysate plus in vitro–translated Vg1RBP/vera-FLAG in the absence (lane 6) or presence (lane 7) of RNase A. Total (lane 1) and bound (lanes 2–7) proteins were separated by 10% SDS-PAGE and immunoblotted with anti-hnRNP I. (C) Recombinant Vg1RBP/vera and hnRNP I were translated in vitro and mixed; shown in lane 1 is the input amount of in vitro–translated hnRNP I. Immunoprecipitation reactions were performed using protein G beads in the presence of recombinant Vg1RBP/vera and hnRNP I (lane 2) and anti-Vg1RBP/vera bound to protein G beads either alone (lane 3) or with recombinant Vg1RBP/vera and hnRNP I (lane 4). Total (lane 1) and bound (lanes 2–4) proteins were separated by 10% SDS-PAGE, and were immunoblotted with anti-hnRNP I. For each panel (A–C), samples were run on the same gel, but lane order was changed for presentation in the figure; adjacent lanes are boxed.
Figure Legend Snippet: Vg1RBP/vera and hnRNP I interact in vitro and in vivo. (A) Anti-hnRNP I was used to immunoprecipitate endogenous hnRNP I from oocyte S10 lysate. Bound Vg1RBP/vera was detected by immunoblotting (lane 4). Shown in lane 1 is the total Vg1RBP/vera in the lysate, and immunoprecipitation with protein G beads with or without lysate is shown in lanes 2 and 3, respectively. (B) In vitro–translated Vg1RBP/vera-FLAG was incubated with oocyte S10 lysate. Immunoprecipitations were performed using anti-FLAG beads with S10 lysate (lane 2), anti-FLAG beads with in vitro–translated Vg1RBP/vera-FLAG (lane 3), Sepharose beads with S10 lysate plus in vitro–translated Vg1RBP/vera-FLAG (lane 4), anti-FLAG beads alone (lane 5), and anti-FLAG beads with S10 lysate plus in vitro–translated Vg1RBP/vera-FLAG in the absence (lane 6) or presence (lane 7) of RNase A. Total (lane 1) and bound (lanes 2–7) proteins were separated by 10% SDS-PAGE and immunoblotted with anti-hnRNP I. (C) Recombinant Vg1RBP/vera and hnRNP I were translated in vitro and mixed; shown in lane 1 is the input amount of in vitro–translated hnRNP I. Immunoprecipitation reactions were performed using protein G beads in the presence of recombinant Vg1RBP/vera and hnRNP I (lane 2) and anti-Vg1RBP/vera bound to protein G beads either alone (lane 3) or with recombinant Vg1RBP/vera and hnRNP I (lane 4). Total (lane 1) and bound (lanes 2–4) proteins were separated by 10% SDS-PAGE, and were immunoblotted with anti-hnRNP I. For each panel (A–C), samples were run on the same gel, but lane order was changed for presentation in the figure; adjacent lanes are boxed.

Techniques Used: In Vitro, In Vivo, Immunoprecipitation, Incubation, SDS Page, Recombinant

Vg1RBP/vera and hnRNP I interact in both the nucleus and cytoplasm. (A) Vg1RBP/vera-FLAG was expressed in stage III/IV oocytes, and nuclear (lanes 1 and 3–6) or cytoplasmic (lanes 2 and 7–10) lysates were prepared. Immunoprecipitations were performed using Sepharose beads plus Vg1RBP/vera S10 lysates (lanes 3 and 7), anti-FLAG beads alone (lanes 4 and 8), anti-FLAG beads with Vg1RBP/vera-FLAG, and nuclear (lanes 5 and 6) or cytoplasmic (lanes 9 and 10) S10 lysate in the presence (lanes 6 and 10) or absence (lanes 5 and 9) of RNase A. Total (lanes 1 and 2) and bound (lanes 3–10) proteins were separated by 10% SDS-PAGE and immunoblotted with anti-hnRNP I. All samples were run on the same gel, but lane order was changed for presentation in the figure, with adjacent lanes boxed together. (B) Nuclear (lanes 1 and 2) and cytoplasmic (lanes 3 and 4) S10 lysates were prepared and were subjected to digestion with RNase A (lanes 2 and 4) or mock digestion (lanes 1 and 3). Vg1 (top) and VegT (bottom) RNAs present in the lysates were detected by RT-PCR. All samples were run on the same gel; adjacent lanes are grouped together. (C) Nuclear (lanes 1 and 3–6) and cytoplasmic (lanes 2 and 7–10) lysates were prepared from oocytes expressing hnRNP I-FLAG. Immunoprecipitations were performed using Sepharose beads plus lysate (lanes 3 and 7), anti-FLAG beads alone (lanes 4 and 8), and anti-FLAG beads and nuclear (lanes 5 and 6) or cytoplasmic (lanes 9 and 10) S10 lysate either with (lanes 6 and 10) or without (lanes 5 and 9) RNase A treatment. Total (lanes 1 and 2) and bound (lanes 3–10) proteins were separated by SDS-PAGE. Bound Vg1RBP/vera was detected by immunoblot with anti-Vg1RBP/vera (top panels), and immunoprecipitated hnRNP I was detected by immunoblot with anti-hnRNP I (bottom panels). All samples were run on the same gel, but lane order was changed for presentation in the figure, with adjacent lanes boxed together.
Figure Legend Snippet: Vg1RBP/vera and hnRNP I interact in both the nucleus and cytoplasm. (A) Vg1RBP/vera-FLAG was expressed in stage III/IV oocytes, and nuclear (lanes 1 and 3–6) or cytoplasmic (lanes 2 and 7–10) lysates were prepared. Immunoprecipitations were performed using Sepharose beads plus Vg1RBP/vera S10 lysates (lanes 3 and 7), anti-FLAG beads alone (lanes 4 and 8), anti-FLAG beads with Vg1RBP/vera-FLAG, and nuclear (lanes 5 and 6) or cytoplasmic (lanes 9 and 10) S10 lysate in the presence (lanes 6 and 10) or absence (lanes 5 and 9) of RNase A. Total (lanes 1 and 2) and bound (lanes 3–10) proteins were separated by 10% SDS-PAGE and immunoblotted with anti-hnRNP I. All samples were run on the same gel, but lane order was changed for presentation in the figure, with adjacent lanes boxed together. (B) Nuclear (lanes 1 and 2) and cytoplasmic (lanes 3 and 4) S10 lysates were prepared and were subjected to digestion with RNase A (lanes 2 and 4) or mock digestion (lanes 1 and 3). Vg1 (top) and VegT (bottom) RNAs present in the lysates were detected by RT-PCR. All samples were run on the same gel; adjacent lanes are grouped together. (C) Nuclear (lanes 1 and 3–6) and cytoplasmic (lanes 2 and 7–10) lysates were prepared from oocytes expressing hnRNP I-FLAG. Immunoprecipitations were performed using Sepharose beads plus lysate (lanes 3 and 7), anti-FLAG beads alone (lanes 4 and 8), and anti-FLAG beads and nuclear (lanes 5 and 6) or cytoplasmic (lanes 9 and 10) S10 lysate either with (lanes 6 and 10) or without (lanes 5 and 9) RNase A treatment. Total (lanes 1 and 2) and bound (lanes 3–10) proteins were separated by SDS-PAGE. Bound Vg1RBP/vera was detected by immunoblot with anti-Vg1RBP/vera (top panels), and immunoprecipitated hnRNP I was detected by immunoblot with anti-hnRNP I (bottom panels). All samples were run on the same gel, but lane order was changed for presentation in the figure, with adjacent lanes boxed together.

Techniques Used: SDS Page, Reverse Transcription Polymerase Chain Reaction, Expressing, Immunoprecipitation

41) Product Images from "16S rRNA Is Bound to Era of Streptococcus pneumoniae"

Article Title: 16S rRNA Is Bound to Era of Streptococcus pneumoniae

Journal: Journal of Bacteriology

doi:

Analysis of RNA association with the Era protein of  S. pneumoniae. E. coli  rRNAs were isolated as described in Materials and Methods. All samples were electrophoresed on a 1.5% agarose gel containing ethidium bromide. Lane 1, DNA standards (100-bp increments from bottom to top; Gibco BRL); lanes 2 and 3,  E. coli  rRNA untreated and treated with RNase A, respectively; lanes 4 and 5, phenol-chloroform-extracted material from a purified GST-Era protein preparation untreated and treated with RNase A, respectively; lanes 6 and 7, a purified GST-Era protein preparation untreated and treated with RNase A, respectively; lane 8, phenol-chloroform-extracted material from a purified GST-Era protein preparation treated with DNase.
Figure Legend Snippet: Analysis of RNA association with the Era protein of S. pneumoniae. E. coli rRNAs were isolated as described in Materials and Methods. All samples were electrophoresed on a 1.5% agarose gel containing ethidium bromide. Lane 1, DNA standards (100-bp increments from bottom to top; Gibco BRL); lanes 2 and 3, E. coli rRNA untreated and treated with RNase A, respectively; lanes 4 and 5, phenol-chloroform-extracted material from a purified GST-Era protein preparation untreated and treated with RNase A, respectively; lanes 6 and 7, a purified GST-Era protein preparation untreated and treated with RNase A, respectively; lane 8, phenol-chloroform-extracted material from a purified GST-Era protein preparation treated with DNase.

Techniques Used: Isolation, Agarose Gel Electrophoresis, Purification

Analysis of Era-RNA complex formation in a crude extract of S. pneumoniae by gel filtration column chromatography. A crude extract of S. pneumoniae was prepared, untreated (A) or treated with RNase A (1 mg/ml) (B), and subjected to chromatography on a gel filtration column as described in Materials and Methods. The presence of Era in the fractions collected was detected by Western blotting analysis with polyclonal antibodies prepared against the native Era protein of S. pneumoniae ). The intensity of each band was quantified by scanning as described in Materials and Methods. Lanes 1 and 11, molecular mass markers and purified Era of S. pneumoniae (30 ng), respectively; lanes 2 to 10, fractions 23 to 31, respectively; lanes 12 to 19, fractions 39 to 46, respectively.
Figure Legend Snippet: Analysis of Era-RNA complex formation in a crude extract of S. pneumoniae by gel filtration column chromatography. A crude extract of S. pneumoniae was prepared, untreated (A) or treated with RNase A (1 mg/ml) (B), and subjected to chromatography on a gel filtration column as described in Materials and Methods. The presence of Era in the fractions collected was detected by Western blotting analysis with polyclonal antibodies prepared against the native Era protein of S. pneumoniae ). The intensity of each band was quantified by scanning as described in Materials and Methods. Lanes 1 and 11, molecular mass markers and purified Era of S. pneumoniae (30 ng), respectively; lanes 2 to 10, fractions 23 to 31, respectively; lanes 12 to 19, fractions 39 to 46, respectively.

Techniques Used: Filtration, Column Chromatography, Chromatography, Western Blot, Purification

Effects of RNase A and DNase I treatments on the GST-Era GTPase activity of S. pneumoniae . Purified GST-Era protein (200 μg/ml) was treated with RNase A (200 or 800 μg/ml) or DNase I (100 μg/ml) at 37°C for 30 min. The RNase A- or DNase I-treated and untreated (control) GST-Era proteins (10 μg each) were then tested for their GTPase activities at 37°C for 30 min by the HPLC method as described in Materials and Methods. After the RNase A and DNase I treatments, parts of the GST-Era preparations were also analyzed by agarose gel electrophoresis (see Materials and Methods), and RNA was not detectable by ethidium bromide staining (data not shown).
Figure Legend Snippet: Effects of RNase A and DNase I treatments on the GST-Era GTPase activity of S. pneumoniae . Purified GST-Era protein (200 μg/ml) was treated with RNase A (200 or 800 μg/ml) or DNase I (100 μg/ml) at 37°C for 30 min. The RNase A- or DNase I-treated and untreated (control) GST-Era proteins (10 μg each) were then tested for their GTPase activities at 37°C for 30 min by the HPLC method as described in Materials and Methods. After the RNase A and DNase I treatments, parts of the GST-Era preparations were also analyzed by agarose gel electrophoresis (see Materials and Methods), and RNA was not detectable by ethidium bromide staining (data not shown).

Techniques Used: Activity Assay, Purification, High Performance Liquid Chromatography, Agarose Gel Electrophoresis, Staining

42) Product Images from "CERKL, a Retinal Disease Gene, Encodes an mRNA-Binding Protein That Localizes in Compact and Untranslated mRNPs Associated with Microtubules"

Article Title: CERKL, a Retinal Disease Gene, Encodes an mRNA-Binding Protein That Localizes in Compact and Untranslated mRNPs Associated with Microtubules

Journal: PLoS ONE

doi: 10.1371/journal.pone.0087898

In the compact mRNPs, CERKL interacts with PABP, HSP70 and RPS3 in an mRNA-dependent manner. A and B ) HEK-293T cells were transfected with CERKL-WT ( A ) or with CERKL-C125W mutant ( B ). After 48 h, cells were treated with 100 µg/ml cycloheximide and lysates were treated or not with RNase A (100 µg/mL) in the presence of three different concentrations of NaCl (150, 450 and 600 mM, as indicated). Then, the lysates were immunoprecipitated with anti-Flag M2 affinity beads. The co-immunoprecipitated proteins were analyzed by immunoblot using antibodies that recognize eIF3B (only in A ), PABP, HSP70, RPS3 and Flag (to detect CERKL-WT and its C125W mutant). In the histograms below, the bands corresponding to the various proteins recovered after the Flag IP, in the presence of RNase A, were quantified with respect to CERKL-WT ( A ) or CERKL-C125W ( B ) in each lane. Values are the mean from 4 different experiments. Stars indicate statistically significant differences from the values in the presence of 150 mM NaCl (*p
Figure Legend Snippet: In the compact mRNPs, CERKL interacts with PABP, HSP70 and RPS3 in an mRNA-dependent manner. A and B ) HEK-293T cells were transfected with CERKL-WT ( A ) or with CERKL-C125W mutant ( B ). After 48 h, cells were treated with 100 µg/ml cycloheximide and lysates were treated or not with RNase A (100 µg/mL) in the presence of three different concentrations of NaCl (150, 450 and 600 mM, as indicated). Then, the lysates were immunoprecipitated with anti-Flag M2 affinity beads. The co-immunoprecipitated proteins were analyzed by immunoblot using antibodies that recognize eIF3B (only in A ), PABP, HSP70, RPS3 and Flag (to detect CERKL-WT and its C125W mutant). In the histograms below, the bands corresponding to the various proteins recovered after the Flag IP, in the presence of RNase A, were quantified with respect to CERKL-WT ( A ) or CERKL-C125W ( B ) in each lane. Values are the mean from 4 different experiments. Stars indicate statistically significant differences from the values in the presence of 150 mM NaCl (*p

Techniques Used: Transfection, Mutagenesis, Immunoprecipitation

The compact CERKL-mRNP complexes interact with microtubules. HEK-293T cells were transfected with CERKL-WT and after 48 h a cytoskeletal fraction was isolated from the cells as described in Materials and Methods. Fractions were treated with 15 mM EDTA ( A ) or 100 µg/ml RNase A ( B ) in the presence of 150, 450 and 600 mM NaCl, as shown in the figure, and then subjected to immunoprecipitation with anti-Flag M2 affinity beads. The co-immunoprecipitated proteins were analyzed by immunoblot using antibodies that recognize eIF3B, PABP, HSP70, RPS3 and Flag (for CERKL). In the histograms below, the bands corresponding to the various proteins recovered after the Flag IP, in the presence of RNase A, were quantified with respect to CERKL-WT ( A ) or to CERKL-C125W ( B ) in each lane. Values are the mean from 4 different experiments. Stars indicate statistically significant differences from the values in the presence of 150 mM NaCl (*p
Figure Legend Snippet: The compact CERKL-mRNP complexes interact with microtubules. HEK-293T cells were transfected with CERKL-WT and after 48 h a cytoskeletal fraction was isolated from the cells as described in Materials and Methods. Fractions were treated with 15 mM EDTA ( A ) or 100 µg/ml RNase A ( B ) in the presence of 150, 450 and 600 mM NaCl, as shown in the figure, and then subjected to immunoprecipitation with anti-Flag M2 affinity beads. The co-immunoprecipitated proteins were analyzed by immunoblot using antibodies that recognize eIF3B, PABP, HSP70, RPS3 and Flag (for CERKL). In the histograms below, the bands corresponding to the various proteins recovered after the Flag IP, in the presence of RNase A, were quantified with respect to CERKL-WT ( A ) or to CERKL-C125W ( B ) in each lane. Values are the mean from 4 different experiments. Stars indicate statistically significant differences from the values in the presence of 150 mM NaCl (*p

Techniques Used: Transfection, Isolation, Immunoprecipitation

CERKL localizes to polysomes and compact mRNPs. A ) HeLa cells stably expressing CERKL WT-HA or an N-terminal fragment of the protein (CERKL 1-256-HA), generated as described in the Materials and Methods section, were lysed and polysomes were isolated by centrifugation in a 0.5-1.3-1.7-2.1 M discontinuous sucrose gradient. Seven 1 ml fractions (1–7) and the pellet (P) were analyzed by immunoblot using antibodies that recognize HA, the ribosomal protein RPS6. Actin was used as control. B and C ) Similar experiments were carried out incubating the cell lysates with EDTA (15 mM) ( B ) or RNase A (100 µg/mL) ( C ) before centrifugation. Antibodies that recognize HA and the ribosomal proteins RPS6 and RPL26 were used. D – F ) The pellet ( D ) and fractions 2 and 3 of the gradient ( E and F ) were treated with 25 mM ( D and E ) or 450 mM ( F ) NaCl, with or without RNase A (100 µg/mL) and subsequently centrifuged in a continuous sucrose density gradient (0.3–1.5 M) as described in Materials and Methods. The new fractions (1–11, in D and 1–10, in E and F ) and the pellet (P) were immunoblotted with HA and RPS6 ( D ) or RNase A ( E and F ) antibodies. Below the Western blots, the concentration of RNA quantified at 260 nm is shown. G ) The same experiment as in E and F , with fractions 2 and 3 treated with 30 mM EDTA or 1 mM puromycin at 25 mM and 450 mM NaCl before the continuous sucrose gradient. Ten fractions and the pellet were analyzed by immunoblot using antibodies that recognize HA.
Figure Legend Snippet: CERKL localizes to polysomes and compact mRNPs. A ) HeLa cells stably expressing CERKL WT-HA or an N-terminal fragment of the protein (CERKL 1-256-HA), generated as described in the Materials and Methods section, were lysed and polysomes were isolated by centrifugation in a 0.5-1.3-1.7-2.1 M discontinuous sucrose gradient. Seven 1 ml fractions (1–7) and the pellet (P) were analyzed by immunoblot using antibodies that recognize HA, the ribosomal protein RPS6. Actin was used as control. B and C ) Similar experiments were carried out incubating the cell lysates with EDTA (15 mM) ( B ) or RNase A (100 µg/mL) ( C ) before centrifugation. Antibodies that recognize HA and the ribosomal proteins RPS6 and RPL26 were used. D – F ) The pellet ( D ) and fractions 2 and 3 of the gradient ( E and F ) were treated with 25 mM ( D and E ) or 450 mM ( F ) NaCl, with or without RNase A (100 µg/mL) and subsequently centrifuged in a continuous sucrose density gradient (0.3–1.5 M) as described in Materials and Methods. The new fractions (1–11, in D and 1–10, in E and F ) and the pellet (P) were immunoblotted with HA and RPS6 ( D ) or RNase A ( E and F ) antibodies. Below the Western blots, the concentration of RNA quantified at 260 nm is shown. G ) The same experiment as in E and F , with fractions 2 and 3 treated with 30 mM EDTA or 1 mM puromycin at 25 mM and 450 mM NaCl before the continuous sucrose gradient. Ten fractions and the pellet were analyzed by immunoblot using antibodies that recognize HA.

Techniques Used: Stable Transfection, Expressing, Generated, Isolation, Centrifugation, Western Blot, Concentration Assay

43) Product Images from "The first crystal structure of human RNase 6 reveals a novel substrate-binding and cleavage site arrangement"

Article Title: The first crystal structure of human RNase 6 reveals a novel substrate-binding and cleavage site arrangement

Journal: Biochemical Journal

doi: 10.1042/BCJ20160245

Predicted RNase 6 and RNase A structures in complex with dinucleotides Predicted structure of RNase 6 and RNase A in complex with CpA, UpA and UpG dinucleotides after MD simulations, as detailed in the Materials and methods section. Nucleotides are coloured green. RNases interacting residues are coloured magenta. Hydrogen bonds are coloured yellow. Structures were drawn with UCSF Chimera 1.10 [ 77 ].
Figure Legend Snippet: Predicted RNase 6 and RNase A structures in complex with dinucleotides Predicted structure of RNase 6 and RNase A in complex with CpA, UpA and UpG dinucleotides after MD simulations, as detailed in the Materials and methods section. Nucleotides are coloured green. RNases interacting residues are coloured magenta. Hydrogen bonds are coloured yellow. Structures were drawn with UCSF Chimera 1.10 [ 77 ].

Techniques Used:

Analysis of polynucleotide cleavage pattern by RNase 6 and RNase A Poly(C) cleavage pattern obtained by RNase 6 ( A ) compared with RNase A [ 27 ] ( B ). Chromatography profiles of poly(C) digestion products are shown at selected incubation times corresponding to representative steps of the catalysis process. See the Materials and methods section for substrate digestion conditions.
Figure Legend Snippet: Analysis of polynucleotide cleavage pattern by RNase 6 and RNase A Poly(C) cleavage pattern obtained by RNase 6 ( A ) compared with RNase A [ 27 ] ( B ). Chromatography profiles of poly(C) digestion products are shown at selected incubation times corresponding to representative steps of the catalysis process. See the Materials and methods section for substrate digestion conditions.

Techniques Used: Chromatography, Incubation

Primary structure of human RNases and 3D structure of RNase 6 coloured by residue conservation score ( A ) Structure-based sequence of the eight canonical human RNases together with RNase A. The active sites are highlighted in yellow. The four disulfide bonds are labelled with green numbers. Tested mutations on RNase 6 are indicated with red arrows. The alignment was performed using ClustalW, and drawn using ESPript ( http://espript.ibcp.fr/ESPript/ ). Labels are as follows: red box, white character for strict identity; red character for similarity in a group and character with a blue frame for similarity across groups. ( B ) RNase 6 3D structure surface representation using the CONSURF web server ( http://consurf.tau.ac.il/ ) featuring the relationships among the evolutionary conservation of amino acid positions within the RNase A family. The 3D structure shows residues coloured by their conservation score using the colour-coding bar at the bottom. Sulfate anions (S1–S4) and the glycerol (GOL) molecule found in the crystal structure are depicted. Conserved residues belonging to the RNase catalytic site and interacting with bound sulfate anions are labelled.
Figure Legend Snippet: Primary structure of human RNases and 3D structure of RNase 6 coloured by residue conservation score ( A ) Structure-based sequence of the eight canonical human RNases together with RNase A. The active sites are highlighted in yellow. The four disulfide bonds are labelled with green numbers. Tested mutations on RNase 6 are indicated with red arrows. The alignment was performed using ClustalW, and drawn using ESPript ( http://espript.ibcp.fr/ESPript/ ). Labels are as follows: red box, white character for strict identity; red character for similarity in a group and character with a blue frame for similarity across groups. ( B ) RNase 6 3D structure surface representation using the CONSURF web server ( http://consurf.tau.ac.il/ ) featuring the relationships among the evolutionary conservation of amino acid positions within the RNase A family. The 3D structure shows residues coloured by their conservation score using the colour-coding bar at the bottom. Sulfate anions (S1–S4) and the glycerol (GOL) molecule found in the crystal structure are depicted. Conserved residues belonging to the RNase catalytic site and interacting with bound sulfate anions are labelled.

Techniques Used: Sequencing

44) Product Images from "The RNA helicase Ddx5/p68 binds to hUpf3 and enhances NMD of Ddx17/p72 and Smg5 mRNA"

Article Title: The RNA helicase Ddx5/p68 binds to hUpf3 and enhances NMD of Ddx17/p72 and Smg5 mRNA

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkt538

Physical interaction of Ddx5 and hUpf3B. ( A ) Binding of Ddx5 to the C-terminal part of Upf3B. HeLa cells were transfected with plasmids encoding FLAG-tagged Upf3B or one of the Upf3B deletion mutants (FLAG-Upf3B 1–270 or FLAG-Upf3B 270–470 ) or with an empty vector (vector) for 48 h. Cells were lysed in the presence of RNase A, immunoprecipitated with anti-FLAG antibodies and analyzed for indicated proteins by western blotting. ( B ) Binding of Upf3B to the N-terminal part of Ddx5. Left panel, HeLa cells were transfected with GFP-Upf1 for 48 h, and cell lysates were immunoprecipitated with anti-GFP antibodies followed by western blot analysis of indicated proteins. Right panel, HeLa cells were transfected with plasmids encoding FLAG-tagged Upf3B and one of the KT3-tagged Ddx5 deletion mutants (Ddx5-KT3 1–189 or Ddx5-KT3 190–614 ). Pull-down assays were performed with anti-FLAG-antibodies and Ddx5-KT3 deletion mutants as the prey-proteins (labeled by asterisks) in the presence of RNase A followed by western blot analysis with anti-Upf3B and anti-KT3 antibodies.
Figure Legend Snippet: Physical interaction of Ddx5 and hUpf3B. ( A ) Binding of Ddx5 to the C-terminal part of Upf3B. HeLa cells were transfected with plasmids encoding FLAG-tagged Upf3B or one of the Upf3B deletion mutants (FLAG-Upf3B 1–270 or FLAG-Upf3B 270–470 ) or with an empty vector (vector) for 48 h. Cells were lysed in the presence of RNase A, immunoprecipitated with anti-FLAG antibodies and analyzed for indicated proteins by western blotting. ( B ) Binding of Upf3B to the N-terminal part of Ddx5. Left panel, HeLa cells were transfected with GFP-Upf1 for 48 h, and cell lysates were immunoprecipitated with anti-GFP antibodies followed by western blot analysis of indicated proteins. Right panel, HeLa cells were transfected with plasmids encoding FLAG-tagged Upf3B and one of the KT3-tagged Ddx5 deletion mutants (Ddx5-KT3 1–189 or Ddx5-KT3 190–614 ). Pull-down assays were performed with anti-FLAG-antibodies and Ddx5-KT3 deletion mutants as the prey-proteins (labeled by asterisks) in the presence of RNase A followed by western blot analysis with anti-Upf3B and anti-KT3 antibodies.

Techniques Used: Binding Assay, Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Labeling

Interaction of Ddx5 with human NMD key factors and mRNPs. ( A ) Co-IP of human Upf1, Upf2 and Upf3 with Ddx5. HeLa cell lysates were immunopreciptated with an anti-Ddx5-antibody (C10) or a control antibody (PAb101) in the presence (+) or absence (−) of RNase A at different NaCl concentrations. Ddx5, Upf1, Upf2 and Upf3 were detected with respective antibodies by western blotting. ( B ) Co-IP of mRNP components with Ddx5. HeLa cells were subjected to IP using an anti-Ddx5 antibody (C10) or no antibody as a control (− antib.) in the presence (+) or absence (−) of RNase A. Immunopurified proteins were analyzed by western blotting with respective antibodies. Asterisks mark mouse IgG light chains stained by the secondary antibody because of their cross-reactivity. Input analysis without RNase A essentially gave the same result as that with RNase A and is not shown. ( C ) Co-IP of Ddx5 with CBP80. HeLa cells were subjected to IP using an anti-CBP80 antibody or no antibody (− antib.) in the presence (+) or absence (−) of RNase A. Immunoprecipitated proteins were analyzed by western blotting with respective antibodies. ( D ) Control of RNase A digestion of cell extracts used after indicated times by agarose gel electrophoresis. We show the EthBr staining of the gel with marked 28 S and 18 S rRNA. ( E ) Co-localization of Ddx5 and Upf1 (left panel) and Ddx5 and Ago2 (right panel) in HeLa cells. HeLa cells co-transfected with vectors encoding Ddx5-KT3 (red) and Upf1-GFP (green) or Ddx5-KT3 (green) and Ago2-HA (red) were analyzed by immunofluorescence microscopy. Nuclei were stained with DAPI (blue). White arrows indicate co-localization of Ddx5 with Upf1 and Ago2, respectively, in cytoplasmic granules.
Figure Legend Snippet: Interaction of Ddx5 with human NMD key factors and mRNPs. ( A ) Co-IP of human Upf1, Upf2 and Upf3 with Ddx5. HeLa cell lysates were immunopreciptated with an anti-Ddx5-antibody (C10) or a control antibody (PAb101) in the presence (+) or absence (−) of RNase A at different NaCl concentrations. Ddx5, Upf1, Upf2 and Upf3 were detected with respective antibodies by western blotting. ( B ) Co-IP of mRNP components with Ddx5. HeLa cells were subjected to IP using an anti-Ddx5 antibody (C10) or no antibody as a control (− antib.) in the presence (+) or absence (−) of RNase A. Immunopurified proteins were analyzed by western blotting with respective antibodies. Asterisks mark mouse IgG light chains stained by the secondary antibody because of their cross-reactivity. Input analysis without RNase A essentially gave the same result as that with RNase A and is not shown. ( C ) Co-IP of Ddx5 with CBP80. HeLa cells were subjected to IP using an anti-CBP80 antibody or no antibody (− antib.) in the presence (+) or absence (−) of RNase A. Immunoprecipitated proteins were analyzed by western blotting with respective antibodies. ( D ) Control of RNase A digestion of cell extracts used after indicated times by agarose gel electrophoresis. We show the EthBr staining of the gel with marked 28 S and 18 S rRNA. ( E ) Co-localization of Ddx5 and Upf1 (left panel) and Ddx5 and Ago2 (right panel) in HeLa cells. HeLa cells co-transfected with vectors encoding Ddx5-KT3 (red) and Upf1-GFP (green) or Ddx5-KT3 (green) and Ago2-HA (red) were analyzed by immunofluorescence microscopy. Nuclei were stained with DAPI (blue). White arrows indicate co-localization of Ddx5 with Upf1 and Ago2, respectively, in cytoplasmic granules.

Techniques Used: Co-Immunoprecipitation Assay, Western Blot, Staining, Immunoprecipitation, Agarose Gel Electrophoresis, Transfection, Immunofluorescence, Microscopy

45) Product Images from "The mRNP remodeling mediated by UPF1 promotes rapid degradation of replication-dependent histone mRNA"

Article Title: The mRNP remodeling mediated by UPF1 promotes rapid degradation of replication-dependent histone mRNA

Journal: Nucleic Acids Research

doi: 10.1093/nar/gku610

PNRC2 and SMG5 associate with SLBP in a way that depends on UPF1 phosphorylation. ( A ) HEK293T cells were transiently transfected with plasmids expressing FLAG-SLBP and Myc-PNRC2. Cells were either treated or not treated with 5 mM HU for 20 min before cell harvest. Total-cell extracts were treated with RNase A and subjected to IP using α-FLAG-conjugated agarose beads. ( B ) HEK293T cells were transiently transfected with either UPF1 siRNA or nonspecific control siRNA. Two days later, cells were retransfected with plasmids expressing FLAG-SLBP and Myc-PNRC2. Cells were treated with HU for 20 min before cell harvest. Total-cell extracts were treated with RNase A and subjected to IP using α-FLAG-conjugated agarose beads. The levels of co-immunopurified proteins were normalized to the levels of immunopurified FLAG-SLBP. The normalized levels obtained in the IP of FLAG-SLBP in the presence of control siRNA were arbitrarily set to 1. Each panel of results is representative of at least two independently performed transfections and IPs. ( C ) As performed in Figure 5B , except that HEK293T cells were transfected with plasmids expressing FLAG-SLBP and either Myc-UPF1-WT or Myc-UPF1-HP. Each panel of results is representative of at least two independently performed transfections and IPs.
Figure Legend Snippet: PNRC2 and SMG5 associate with SLBP in a way that depends on UPF1 phosphorylation. ( A ) HEK293T cells were transiently transfected with plasmids expressing FLAG-SLBP and Myc-PNRC2. Cells were either treated or not treated with 5 mM HU for 20 min before cell harvest. Total-cell extracts were treated with RNase A and subjected to IP using α-FLAG-conjugated agarose beads. ( B ) HEK293T cells were transiently transfected with either UPF1 siRNA or nonspecific control siRNA. Two days later, cells were retransfected with plasmids expressing FLAG-SLBP and Myc-PNRC2. Cells were treated with HU for 20 min before cell harvest. Total-cell extracts were treated with RNase A and subjected to IP using α-FLAG-conjugated agarose beads. The levels of co-immunopurified proteins were normalized to the levels of immunopurified FLAG-SLBP. The normalized levels obtained in the IP of FLAG-SLBP in the presence of control siRNA were arbitrarily set to 1. Each panel of results is representative of at least two independently performed transfections and IPs. ( C ) As performed in Figure 5B , except that HEK293T cells were transfected with plasmids expressing FLAG-SLBP and either Myc-UPF1-WT or Myc-UPF1-HP. Each panel of results is representative of at least two independently performed transfections and IPs.

Techniques Used: Transfection, Expressing

ATR and DNA-PK activated upon the inhibition of DNA replication triggers UPF1 phosphorylation and promotes UPF1–SLBP interaction. ( A ) IP of endogenous CTIF in the presence of OA. HeLa cells were treated with either ethanol (0 nM) or OA (75 nM) for 5 h before IP. Total-cell extracts were analyzed either before or after IP using α-CTIF antibody or rIgG. The levels of co-immunopurified SLBP and SLIP1 were normalized to the levels of immunopurified CTIF. The normalized levels obtained in the IP of CTIF with the treatment of ethanol were arbitrarily set to 1. ( B ) IP of FLAG-CTIF in the presence of PIKK inhibitors. HeLa cells transiently expressing FLAG-CTIF were pre-treated with 15 mM caffeine, 20 μM ATM/ATR inhibitor, 140 μM LY294002 or 1.3 μM ATM inhibitor for 2 h before cell harvest. Cells were either untreated or treated with 5 mM HU for 1 h before cell harvest. IPs were performed using α-FLAG antibody. The level of phosphorylated FLAG-CTIF (p-FLAG-CTIF) was determined by western blotting using α-phospho-S/TQ antibody. The results in panels A and B are representative of at least three independently performed transfections and IPs. ( C ) IP of FLAG-UPF1 using extracts of cells treated with either 5 mM HU or HU/alkaline phosphatase (AP). HeLa cells were transiently transfected with plasmid expressing FLAG-UPF1. Cells were either untreated or treated with 5 mM HU for 1 h before cell harvest. IPs were performed using α-FLAG-conjugated agarose beads. The immunopurified complex was or was not treated with AP. The level of phosphorylated FLAG-UPF1 (p-FLAG-UPF1) was determined by western blotting using α-phospho-S/TQ antibody. To show that western blotting used in this study was sufficiently semi-quantitative, 3-fold serial dilutions of total-cell extracts are represented in the four left-most lanes. ( D ) IP of FLAG-UPF1-WT and FLAG-UPF1-HP. HeLa cells were transiently transfected with plasmid expressing either FLAG, FLAG-UPF1-WT or -HP. Total-cell extracts were either untreated or treated with RNase A and subjected to IP using α-FLAG antibody-conjugated agarose beads. The levels of phosphorylated FLAG-UPF1 (p-FLAG-UPF1) were determined by western blotting using α-phospho-S/TQ antibody (top). The levels of p-FLAG-UPF1 and co-immunopurified SLBP were normalized to the levels of immunopurified FLAG-UPF1. The normalized levels obtained in the IP of FLAG-UPF1-WT were arbitrarily set to 1. Complete removal of cellular RNAs by RNase A treatment was confirmed by RT-PCR using α-[ 32 P]-dATP (bottom). To show that the RT-PCR used in this study is sufficiently semi-quantitative, RT-PCR products of 2-fold serially diluted total RNAs were loaded in the four left-most lanes. The results in panels C and D are representative of at least three independently performed transfections and IPs.
Figure Legend Snippet: ATR and DNA-PK activated upon the inhibition of DNA replication triggers UPF1 phosphorylation and promotes UPF1–SLBP interaction. ( A ) IP of endogenous CTIF in the presence of OA. HeLa cells were treated with either ethanol (0 nM) or OA (75 nM) for 5 h before IP. Total-cell extracts were analyzed either before or after IP using α-CTIF antibody or rIgG. The levels of co-immunopurified SLBP and SLIP1 were normalized to the levels of immunopurified CTIF. The normalized levels obtained in the IP of CTIF with the treatment of ethanol were arbitrarily set to 1. ( B ) IP of FLAG-CTIF in the presence of PIKK inhibitors. HeLa cells transiently expressing FLAG-CTIF were pre-treated with 15 mM caffeine, 20 μM ATM/ATR inhibitor, 140 μM LY294002 or 1.3 μM ATM inhibitor for 2 h before cell harvest. Cells were either untreated or treated with 5 mM HU for 1 h before cell harvest. IPs were performed using α-FLAG antibody. The level of phosphorylated FLAG-CTIF (p-FLAG-CTIF) was determined by western blotting using α-phospho-S/TQ antibody. The results in panels A and B are representative of at least three independently performed transfections and IPs. ( C ) IP of FLAG-UPF1 using extracts of cells treated with either 5 mM HU or HU/alkaline phosphatase (AP). HeLa cells were transiently transfected with plasmid expressing FLAG-UPF1. Cells were either untreated or treated with 5 mM HU for 1 h before cell harvest. IPs were performed using α-FLAG-conjugated agarose beads. The immunopurified complex was or was not treated with AP. The level of phosphorylated FLAG-UPF1 (p-FLAG-UPF1) was determined by western blotting using α-phospho-S/TQ antibody. To show that western blotting used in this study was sufficiently semi-quantitative, 3-fold serial dilutions of total-cell extracts are represented in the four left-most lanes. ( D ) IP of FLAG-UPF1-WT and FLAG-UPF1-HP. HeLa cells were transiently transfected with plasmid expressing either FLAG, FLAG-UPF1-WT or -HP. Total-cell extracts were either untreated or treated with RNase A and subjected to IP using α-FLAG antibody-conjugated agarose beads. The levels of phosphorylated FLAG-UPF1 (p-FLAG-UPF1) were determined by western blotting using α-phospho-S/TQ antibody (top). The levels of p-FLAG-UPF1 and co-immunopurified SLBP were normalized to the levels of immunopurified FLAG-UPF1. The normalized levels obtained in the IP of FLAG-UPF1-WT were arbitrarily set to 1. Complete removal of cellular RNAs by RNase A treatment was confirmed by RT-PCR using α-[ 32 P]-dATP (bottom). To show that the RT-PCR used in this study is sufficiently semi-quantitative, RT-PCR products of 2-fold serially diluted total RNAs were loaded in the four left-most lanes. The results in panels C and D are representative of at least three independently performed transfections and IPs.

Techniques Used: Inhibition, Expressing, Western Blot, Transfection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR

46) Product Images from "eIF4GI Facilitates the MicroRNA-Mediated Gene Silencing"

Article Title: eIF4GI Facilitates the MicroRNA-Mediated Gene Silencing

Journal: PLoS ONE

doi: 10.1371/journal.pone.0055725

eIF4GI associates with Ago2. ( A ) Schematic diagram of human eIF4GI. ‘4E’ means ‘the eIF4E-binding motif’. Plasmids were constructed for expression of various Flag-tagged eIF4GI fragments in human cells. ( B ) The N-terminal and middle domains of eIF4GI participate in the eIF4GI-Ago2 association. Plasmids expressing Flag-tagged eIF4GI variants and myc-tagged full-length Ago2 were co-transfected in 293FT cells, and their associations were examined by Flag immunoprecipitation (Flag-IP) with the Flag-resin. The levels of Ago2 and the eIF4GI mutants (left panel) and the amount of co-precipitated Ago2 (right panel) were monitored by Western blotting using the indicated antibodies. ( C ) Schematic diagram of the N-terminal constructs of eIF4GI for fine mapping of the region required for the association with Ago2. ( D ) Determination of the Ago2-associated region in eIF4GI. WCEs from 293FT cells expressing myc-tagged full-length Ago2 and N-terminal variants of eIF4GI serially deleted from the C- or N-termini were subjected to Flag-IP. The expressions of Ago2 and the eIF4GI derivatives (lower panel) and the amount of precipitated Ago2 (upper panel) were examined using the indicated antibodies. ( E ) RNA-independent association of Ago2 with eIF4GI (aa 42–202). WCEs from 293FT cells expressing myc-tagged Ago2 and Flag-tagged eIF4GI-NtP were treated with (lanes 2 and 4) and without (lanes 1 and 3) RNase A and subjected to Flag-IP.
Figure Legend Snippet: eIF4GI associates with Ago2. ( A ) Schematic diagram of human eIF4GI. ‘4E’ means ‘the eIF4E-binding motif’. Plasmids were constructed for expression of various Flag-tagged eIF4GI fragments in human cells. ( B ) The N-terminal and middle domains of eIF4GI participate in the eIF4GI-Ago2 association. Plasmids expressing Flag-tagged eIF4GI variants and myc-tagged full-length Ago2 were co-transfected in 293FT cells, and their associations were examined by Flag immunoprecipitation (Flag-IP) with the Flag-resin. The levels of Ago2 and the eIF4GI mutants (left panel) and the amount of co-precipitated Ago2 (right panel) were monitored by Western blotting using the indicated antibodies. ( C ) Schematic diagram of the N-terminal constructs of eIF4GI for fine mapping of the region required for the association with Ago2. ( D ) Determination of the Ago2-associated region in eIF4GI. WCEs from 293FT cells expressing myc-tagged full-length Ago2 and N-terminal variants of eIF4GI serially deleted from the C- or N-termini were subjected to Flag-IP. The expressions of Ago2 and the eIF4GI derivatives (lower panel) and the amount of precipitated Ago2 (upper panel) were examined using the indicated antibodies. ( E ) RNA-independent association of Ago2 with eIF4GI (aa 42–202). WCEs from 293FT cells expressing myc-tagged Ago2 and Flag-tagged eIF4GI-NtP were treated with (lanes 2 and 4) and without (lanes 1 and 3) RNase A and subjected to Flag-IP.

Techniques Used: Binding Assay, Construct, Expressing, Transfection, Immunoprecipitation, Western Blot

Human Ago associates with the cap-binding complex. ( A ) The cap-association of endogenous Ago2 proteins from HeLa cells were examined by a cap-pulldown assay, using 2 mg of whole-cell extracts (WCEs) for incubation with either control-resin (lane 6) or cap-resin in the presence (lane 8) or absence (lane 7) of the cap analog. ( B ) The cap-association of miRNAs. WCEs from HeLa cells (2 mg) were subjected to a cap-pulldown assay, and the cap-associated RNAs were extracted and subjected to UREA-PAGE followed by Northern blotting using radiolabeled probes against the indicated miRNAs. For comparison, various amounts equal to 4.4–0.8% of the total RNAs contained in WCEs used for the cap-pulldown assays (∼10–2 µg each) were loaded in lanes 1–5. ( C ) The cap-associations of ectopically expressed proteins were monitored as in panel A , except for using 2 mg of WCEs from 293FT cells transfected with plasmids expressing Flag-tagged Ago1, Ago2 or Dicer. ( D ) Cap-pulldown assays were done using 2 mg of WCEs from 293FT cells expressing Flag-Ago2 with 200 µM of G(5′)ppp(3′)G (lane 3) or m 7 G(5′)ppp(3′)G (lane 4). ( E ) The RNA-independent cap-association of Ago2. 2 mg of WCEs from 293FT cells ectopically expressing myc-tagged Ago2 were treated with (lanes 2 and 5) or without (lanes 1, 3 and 4) RNase A and subjected to cap-pulldown assays. In panels A and C , various amounts corresponding to 2–0.4% of WCEs used in the pulldown assay were loaded in lanes 1–5 for comparison. In panels A , C , D and E , Western blot analyses were performed using the indicated antibodies.
Figure Legend Snippet: Human Ago associates with the cap-binding complex. ( A ) The cap-association of endogenous Ago2 proteins from HeLa cells were examined by a cap-pulldown assay, using 2 mg of whole-cell extracts (WCEs) for incubation with either control-resin (lane 6) or cap-resin in the presence (lane 8) or absence (lane 7) of the cap analog. ( B ) The cap-association of miRNAs. WCEs from HeLa cells (2 mg) were subjected to a cap-pulldown assay, and the cap-associated RNAs were extracted and subjected to UREA-PAGE followed by Northern blotting using radiolabeled probes against the indicated miRNAs. For comparison, various amounts equal to 4.4–0.8% of the total RNAs contained in WCEs used for the cap-pulldown assays (∼10–2 µg each) were loaded in lanes 1–5. ( C ) The cap-associations of ectopically expressed proteins were monitored as in panel A , except for using 2 mg of WCEs from 293FT cells transfected with plasmids expressing Flag-tagged Ago1, Ago2 or Dicer. ( D ) Cap-pulldown assays were done using 2 mg of WCEs from 293FT cells expressing Flag-Ago2 with 200 µM of G(5′)ppp(3′)G (lane 3) or m 7 G(5′)ppp(3′)G (lane 4). ( E ) The RNA-independent cap-association of Ago2. 2 mg of WCEs from 293FT cells ectopically expressing myc-tagged Ago2 were treated with (lanes 2 and 5) or without (lanes 1, 3 and 4) RNase A and subjected to cap-pulldown assays. In panels A and C , various amounts corresponding to 2–0.4% of WCEs used in the pulldown assay were loaded in lanes 1–5 for comparison. In panels A , C , D and E , Western blot analyses were performed using the indicated antibodies.

Techniques Used: Binding Assay, Incubation, Polyacrylamide Gel Electrophoresis, Northern Blot, Transfection, Expressing, Western Blot

47) Product Images from "Nod-like receptor protein 3 inflammasome activation by Escherichia coli RNA induces transforming growth factor beta 1 secretion in hepatic stellate cells"

Article Title: Nod-like receptor protein 3 inflammasome activation by Escherichia coli RNA induces transforming growth factor beta 1 secretion in hepatic stellate cells

Journal: Bosnian Journal of Basic Medical Sciences

doi: 10.17305/bjbms.2016.699

Interleukin-1 beta (IL-1β) induction by Escherichia coli RNA, (A) E. coli RNA stimulated IL-1β secretion by hepatic stellate cells (HSC)-T6 cells, which was abrogated upon RNase A digestion, (B) IL-1β expression was elevated, whereas pro-IL-1β expression was reduced in HSC-T6 cells after E. coli RNA transfection. * p
Figure Legend Snippet: Interleukin-1 beta (IL-1β) induction by Escherichia coli RNA, (A) E. coli RNA stimulated IL-1β secretion by hepatic stellate cells (HSC)-T6 cells, which was abrogated upon RNase A digestion, (B) IL-1β expression was elevated, whereas pro-IL-1β expression was reduced in HSC-T6 cells after E. coli RNA transfection. * p

Techniques Used: Expressing, Transfection

Induction of transforming growth factor beta 1 (TGF-β1) and other pro-fibrogenic factors by Escherichia coli RNA, (A) E. coli RNA stimulated TGF-β1 secretion by hepatic stellate cells (HSC)-T6 cells, which was abolished upon RNase A digestion, (B-D) E. coli RNA induced expression of for α-smooth muscle actin, collagen Type I α1, tissue inhibitor of metalloproteinases 1 in HSC-T6 cells, respectively. * p
Figure Legend Snippet: Induction of transforming growth factor beta 1 (TGF-β1) and other pro-fibrogenic factors by Escherichia coli RNA, (A) E. coli RNA stimulated TGF-β1 secretion by hepatic stellate cells (HSC)-T6 cells, which was abolished upon RNase A digestion, (B-D) E. coli RNA induced expression of for α-smooth muscle actin, collagen Type I α1, tissue inhibitor of metalloproteinases 1 in HSC-T6 cells, respectively. * p

Techniques Used: Expressing

48) Product Images from "Long-living RNA in the CNS of terrestrial snail"

Article Title: Long-living RNA in the CNS of terrestrial snail

Journal: RNA Biology

doi: 10.1080/15476286.2017.1411460

A – Click-iT reaction in the section of right cerebral ganglion of an adult animal after 4 hrs of isolated CNS incubation in the 1 mM EU solution. B- schemata of the right cerebral ganglion. PC – procerebral lobe, MsC – mesocerebral lobe, MtC – metacerebral lobe. C – early staining reaction in the procerebral lobe of juvenile animal after 1 hr of isolated CNS incubation in 1 mM EU solution (right cerebral ganglion, section). F – BrdU-immunopositive neurons in 2 days after the whole animal immersion in the BrdU solution. Note the spatial coincidence of reaction product in C and F. D, and G: parallel sections of procerebral lobe in adult brain, untreated (D), and treated (G) with RNase A before the EU visualization. E, and H: nucleolar and nuclear EU staining in the giant neuron of adult snail after incubation of CNS for 16 hrs in 1 mM EU. E, and H – the same section stained consequently with Alexa 488-azide (E) and Hoechst (H). Note the dark spots in H at the place of nucleoli. Scale bars: 500 μm (A, and B), 100 μm (C, D, F, G), 25 μm (E, and H).
Figure Legend Snippet: A – Click-iT reaction in the section of right cerebral ganglion of an adult animal after 4 hrs of isolated CNS incubation in the 1 mM EU solution. B- schemata of the right cerebral ganglion. PC – procerebral lobe, MsC – mesocerebral lobe, MtC – metacerebral lobe. C – early staining reaction in the procerebral lobe of juvenile animal after 1 hr of isolated CNS incubation in 1 mM EU solution (right cerebral ganglion, section). F – BrdU-immunopositive neurons in 2 days after the whole animal immersion in the BrdU solution. Note the spatial coincidence of reaction product in C and F. D, and G: parallel sections of procerebral lobe in adult brain, untreated (D), and treated (G) with RNase A before the EU visualization. E, and H: nucleolar and nuclear EU staining in the giant neuron of adult snail after incubation of CNS for 16 hrs in 1 mM EU. E, and H – the same section stained consequently with Alexa 488-azide (E) and Hoechst (H). Note the dark spots in H at the place of nucleoli. Scale bars: 500 μm (A, and B), 100 μm (C, D, F, G), 25 μm (E, and H).

Techniques Used: Isolation, Incubation, Staining

Click-iT reaction in the CNS of 2-week-old animals after 4 h of incubation in 1mM EU solution. A-B: Influence of DNAse treatment. A, and B – pleuro-parietal (A) and cerebral (B) ganglia sections processed without DNAse treatment. A1, and B1 – parallel sections treated, before processing, with DNAse. C-E: Influence of actinomycin (C), urea (D), and thymidine (E). C-E – right pedal ganglion processed as whole-mount. Only actinomycin removed the staining. F – Influence of RNAse A pretreatment on the Click-iT reaction. Parallel sections of pleuro-parietal ganglia without (F), and with (F1) RNAse A treatment. Scale bar: 100 μm.
Figure Legend Snippet: Click-iT reaction in the CNS of 2-week-old animals after 4 h of incubation in 1mM EU solution. A-B: Influence of DNAse treatment. A, and B – pleuro-parietal (A) and cerebral (B) ganglia sections processed without DNAse treatment. A1, and B1 – parallel sections treated, before processing, with DNAse. C-E: Influence of actinomycin (C), urea (D), and thymidine (E). C-E – right pedal ganglion processed as whole-mount. Only actinomycin removed the staining. F – Influence of RNAse A pretreatment on the Click-iT reaction. Parallel sections of pleuro-parietal ganglia without (F), and with (F1) RNAse A treatment. Scale bar: 100 μm.

Techniques Used: Incubation, Staining

49) Product Images from "The Antiviral and Cancer Genomic DNA Deaminase APOBEC3H Is Regulated by a RNA-Mediated Dimerization Mechanism"

Article Title: The Antiviral and Cancer Genomic DNA Deaminase APOBEC3H Is Regulated by a RNA-Mediated Dimerization Mechanism

Journal: Molecular cell

doi: 10.1016/j.molcel.2017.12.010

DNA Deaminase Activity of APOBEC3H and Derivatives in 293T Extracts ( A and B ) Comparison of the DNA deaminase activities of untagged wild-type (WT) human A3H or the indicated amino acid substitution mutants in 293T extracts +/− RNase A treatment (S, substrate; P, product). Corresponding immunoblots show levels of A3H in cell lysates using a murine mAb (P1D8) or a rabbit pAb (Novus). β-actin is a loading control.
Figure Legend Snippet: DNA Deaminase Activity of APOBEC3H and Derivatives in 293T Extracts ( A and B ) Comparison of the DNA deaminase activities of untagged wild-type (WT) human A3H or the indicated amino acid substitution mutants in 293T extracts +/− RNase A treatment (S, substrate; P, product). Corresponding immunoblots show levels of A3H in cell lysates using a murine mAb (P1D8) or a rabbit pAb (Novus). β-actin is a loading control.

Techniques Used: Activity Assay, Western Blot

RNase A Digestion Enables APOBEC3H Purification and DNA Deaminase Activity ( A ) Coomassie-stained image showing His6-SUMO-A3H recovery from E. coli +/− RNase A treatment. ( B ) DNA deaminase activity of His6-SUMO-A3H in extracts from E. coli +/− RNase A treatment (mean +/− SD; n = 3 experiments; inset gel image shows A3H-mediated conversion of a single-stranded DNA substrate to product, S to P, in the presence of RNase A). Lysate units (μl volumes) were chosen to include reactions with single-hit kinetics. ( C ) DNA deaminase activity of untagged A3H in extracts from 293T cells, with experimental parameters similar to those in panel B.
Figure Legend Snippet: RNase A Digestion Enables APOBEC3H Purification and DNA Deaminase Activity ( A ) Coomassie-stained image showing His6-SUMO-A3H recovery from E. coli +/− RNase A treatment. ( B ) DNA deaminase activity of His6-SUMO-A3H in extracts from E. coli +/− RNase A treatment (mean +/− SD; n = 3 experiments; inset gel image shows A3H-mediated conversion of a single-stranded DNA substrate to product, S to P, in the presence of RNase A). Lysate units (μl volumes) were chosen to include reactions with single-hit kinetics. ( C ) DNA deaminase activity of untagged A3H in extracts from 293T cells, with experimental parameters similar to those in panel B.

Techniques Used: Purification, Activity Assay, Staining

50) Product Images from "RNA-dependent chromatin localization of KDM4D lysine demethylase promotes H3K9me3 demethylation"

Article Title: RNA-dependent chromatin localization of KDM4D lysine demethylase promotes H3K9me3 demethylation

Journal: Nucleic Acids Research

doi: 10.1093/nar/gku1021

RNA interactions with KDM4D N-terminal region are critical for KDM4D association with chromatin and for the demethylation of H3K9me3 mark. (A) KDM4D is enriched at the chromatin-bound fraction. U2OS cells were subjected to biochemical fractionation as previously described ( 22 ). Samples from the different fractions were resolved and immunoblotted using the indicated antibodies. (B) RNase A treatment disrupts the KDM4D localization at the NP-40-resistant fraction. Mock and RNase A-treated cells were subjected to biochemical fractionation using NP-40 lysis buffer and protein samples were resolved and immunoblotted using the indicated antibodies. (C) The chromatin localization of KDM4D is not affected by RNase H treatment. U2OS cells were treated with Mock or RNase H and subjected to biochemical fractionation as described in (B). (D and E) FRAP and FLIP analyses show that the 1H4R-HRK mutations lead to a remarkable increase in the mobility of KDM4D in cells. U2OS cells were transfected with constructs encoding EGFP-KDM4D-WT or EGFP-KDM4D-1H4R-HRK and subjected to FRAP (D) or FLIP (E) assay. In (D) the plot shows the relative fluorescence intensity over time at the bleached area, normalized to the pre-bleached levels. In (E) the cells were subjected to continuous bleaching in a particular area and the relative fluorescence at a nearby region was plotted against time. The FRAP and the FLIP results are the averages for 12 different cells and similar results were obtained in two different experiments. (F) Biochemical fractionation shows that, unlike EGFP-KDM4D-WT, EGFP-KDM4D-FL-1H4R-HRK lost its association with chromatin. (G) Shows that overexpression of EGFP-KDM4D-FL-1H4R-HRK has no detectable effect on the overall levels of H3K9me3. U2OS-TetON cells were treated with doxycycline for 24 h to induce the expression of EGFP-KDM4D-FL or EGFP-KDM4D-FL-1H4R-HRK, and protein extracts were prepared using hot-lysis procedure and subjected for western blotting using the indicated antibodies. The intensities of H3K9me3 bands were normalized against the corresponding H3 signal and the ratios are shown at the bottom of the blot. ( H ) Representative cells showing that EGFP-KDM4D-FL-1H4R-HRK is unable to demethylate H3K9me3 mark (red). Cells were transfected with constructs encoding either EGFP-KDM4D-WT or EGFP-KDM4D-1H4R-HRK mutant (green) and subjected to immunofluorescence analysis. Nuclei were stained with DAPI (blue). These results are typical of 3 independent experiments and at least 30 different cells were acquired each time. White arrowheads indicate cell transfected with EGFP-KDM4D-WT (top) and EGFP-KDM4D-FL-1H4R-HRK mutant (bottom). C: cytoplasmic; N: nuclear soluble fraction; Chr: chromatin-bound fraction.
Figure Legend Snippet: RNA interactions with KDM4D N-terminal region are critical for KDM4D association with chromatin and for the demethylation of H3K9me3 mark. (A) KDM4D is enriched at the chromatin-bound fraction. U2OS cells were subjected to biochemical fractionation as previously described ( 22 ). Samples from the different fractions were resolved and immunoblotted using the indicated antibodies. (B) RNase A treatment disrupts the KDM4D localization at the NP-40-resistant fraction. Mock and RNase A-treated cells were subjected to biochemical fractionation using NP-40 lysis buffer and protein samples were resolved and immunoblotted using the indicated antibodies. (C) The chromatin localization of KDM4D is not affected by RNase H treatment. U2OS cells were treated with Mock or RNase H and subjected to biochemical fractionation as described in (B). (D and E) FRAP and FLIP analyses show that the 1H4R-HRK mutations lead to a remarkable increase in the mobility of KDM4D in cells. U2OS cells were transfected with constructs encoding EGFP-KDM4D-WT or EGFP-KDM4D-1H4R-HRK and subjected to FRAP (D) or FLIP (E) assay. In (D) the plot shows the relative fluorescence intensity over time at the bleached area, normalized to the pre-bleached levels. In (E) the cells were subjected to continuous bleaching in a particular area and the relative fluorescence at a nearby region was plotted against time. The FRAP and the FLIP results are the averages for 12 different cells and similar results were obtained in two different experiments. (F) Biochemical fractionation shows that, unlike EGFP-KDM4D-WT, EGFP-KDM4D-FL-1H4R-HRK lost its association with chromatin. (G) Shows that overexpression of EGFP-KDM4D-FL-1H4R-HRK has no detectable effect on the overall levels of H3K9me3. U2OS-TetON cells were treated with doxycycline for 24 h to induce the expression of EGFP-KDM4D-FL or EGFP-KDM4D-FL-1H4R-HRK, and protein extracts were prepared using hot-lysis procedure and subjected for western blotting using the indicated antibodies. The intensities of H3K9me3 bands were normalized against the corresponding H3 signal and the ratios are shown at the bottom of the blot. ( H ) Representative cells showing that EGFP-KDM4D-FL-1H4R-HRK is unable to demethylate H3K9me3 mark (red). Cells were transfected with constructs encoding either EGFP-KDM4D-WT or EGFP-KDM4D-1H4R-HRK mutant (green) and subjected to immunofluorescence analysis. Nuclei were stained with DAPI (blue). These results are typical of 3 independent experiments and at least 30 different cells were acquired each time. White arrowheads indicate cell transfected with EGFP-KDM4D-WT (top) and EGFP-KDM4D-FL-1H4R-HRK mutant (bottom). C: cytoplasmic; N: nuclear soluble fraction; Chr: chromatin-bound fraction.

Techniques Used: Fractionation, Lysis, Transfection, Construct, Fluorescence, Over Expression, Expressing, Western Blot, Mutagenesis, Immunofluorescence, Staining

51) Product Images from "5? exon interactions within the human spliceosome establish a framework for exon junction complex structure and assembly"

Article Title: 5? exon interactions within the human spliceosome establish a framework for exon junction complex structure and assembly

Journal: Genes & Development

doi: 10.1101/gad.1030602

Splicing and cross-linking of benzophenone-containing AdML RNAs. ( Upper  panels) Splicing time courses. RNAs were incubated for the times indicated and separated by denaturing PAGE (7%). Substrate and product structures are indicated above and to the left of each panel. Because pre-mRNAs contained only one  32 P label, only a subset of splicing products are detectable. The appearance of doublets for each substrate and product reflects the presence of unligated 3′ RNAs. ( Lower  panel) Protein cross-linking. After incubation on ice for times indicated, samples were irradiated at 302 nm and digested with RNase A. Molecular mass standards and apparent molecular masses of proteins that cross-linked in a splicing-dependent manner are indicated to the left and right, respectively. Note that lanes  13 – 16  contained somewhat less input RNA, and thus exhibit reduced cross-linking intensities compared to other samples.
Figure Legend Snippet: Splicing and cross-linking of benzophenone-containing AdML RNAs. ( Upper panels) Splicing time courses. RNAs were incubated for the times indicated and separated by denaturing PAGE (7%). Substrate and product structures are indicated above and to the left of each panel. Because pre-mRNAs contained only one 32 P label, only a subset of splicing products are detectable. The appearance of doublets for each substrate and product reflects the presence of unligated 3′ RNAs. ( Lower panel) Protein cross-linking. After incubation on ice for times indicated, samples were irradiated at 302 nm and digested with RNase A. Molecular mass standards and apparent molecular masses of proteins that cross-linked in a splicing-dependent manner are indicated to the left and right, respectively. Note that lanes 13 – 16 contained somewhat less input RNA, and thus exhibit reduced cross-linking intensities compared to other samples.

Techniques Used: Incubation, Polyacrylamide Gel Electrophoresis, Irradiation

Splicing and cross-linking of 4-thioU-containing RNAs. ( A, upper panel) Splicing time courses of AdML-GG and AdML-AG RNAs containing single 4-thioU and 32 P labels at position −24. Splicing precursors, intermediates, and products are indicated to the right. ( Lower panel) Pattern of cross-linked proteins for each time point in upper panel. Molecular mass standards and apparent molecular weights of proteins cross-linking in a splicing-specific manner are indicated to the left and right, respectively. ( B ) Effects of nonspecific competitor RNA on cross-linking pattern for AdML-AG RNA. ( Left panel) Following a 60-min incubation under splicing conditions, excess cold competitor RNA was added and reactions incubated for an additional 10 min before UV irradiation and RNase digestion. Open and closed arrows indicate bands that were sensitive or not, respectively, to addition of cold competitor RNA. ( Right panel) Denaturing PAGE (15%) of splicing precursors and products in the presence (+) and absence (−) of cold competitor RNA showing that cross-linking differences in the left panel do not simply reflect a difference in the extent of splicing. ( C ) Glycerol gradient fractionation of splicing reactions containing site-specifically modified AdML-GG (lanes 1 – 7 ) or AdML-AG (lanes 9 – 15 ) substrates. Substrates were incubated for 90 min (AdML-GG) or 120 min (AdML-AG) and irradiated on ice prior to being loaded onto a 10%–30% glycerol gradient. Following glycerol gradient fractionation, fractions (numbers at top) were divided and either resolved by denaturing PAGE ( upper panels) or RNase A digested and separated by SDS-PAGE ( lower panels). Lane 8 corresponds to AdML control mRNA incubated for 45 min under splicing conditions, irradiated on ice, and directly resolved by denaturing PAGE ( upper panels) or RNase A digested and separated by SDS-PAGE ( lower panels), without prior glycerol gradient fractionation. Molecular mass markers and apparent molecular masses of splicing specific cross-linked proteins are indicated to the left and right of each gel, respectively. ( D ) Same as C except with site-specifically modified PIP.B control mRNA (lanes 1 – 7 ) and pre-mRNA (lanes 9 – 15 ).
Figure Legend Snippet: Splicing and cross-linking of 4-thioU-containing RNAs. ( A, upper panel) Splicing time courses of AdML-GG and AdML-AG RNAs containing single 4-thioU and 32 P labels at position −24. Splicing precursors, intermediates, and products are indicated to the right. ( Lower panel) Pattern of cross-linked proteins for each time point in upper panel. Molecular mass standards and apparent molecular weights of proteins cross-linking in a splicing-specific manner are indicated to the left and right, respectively. ( B ) Effects of nonspecific competitor RNA on cross-linking pattern for AdML-AG RNA. ( Left panel) Following a 60-min incubation under splicing conditions, excess cold competitor RNA was added and reactions incubated for an additional 10 min before UV irradiation and RNase digestion. Open and closed arrows indicate bands that were sensitive or not, respectively, to addition of cold competitor RNA. ( Right panel) Denaturing PAGE (15%) of splicing precursors and products in the presence (+) and absence (−) of cold competitor RNA showing that cross-linking differences in the left panel do not simply reflect a difference in the extent of splicing. ( C ) Glycerol gradient fractionation of splicing reactions containing site-specifically modified AdML-GG (lanes 1 – 7 ) or AdML-AG (lanes 9 – 15 ) substrates. Substrates were incubated for 90 min (AdML-GG) or 120 min (AdML-AG) and irradiated on ice prior to being loaded onto a 10%–30% glycerol gradient. Following glycerol gradient fractionation, fractions (numbers at top) were divided and either resolved by denaturing PAGE ( upper panels) or RNase A digested and separated by SDS-PAGE ( lower panels). Lane 8 corresponds to AdML control mRNA incubated for 45 min under splicing conditions, irradiated on ice, and directly resolved by denaturing PAGE ( upper panels) or RNase A digested and separated by SDS-PAGE ( lower panels), without prior glycerol gradient fractionation. Molecular mass markers and apparent molecular masses of splicing specific cross-linked proteins are indicated to the left and right of each gel, respectively. ( D ) Same as C except with site-specifically modified PIP.B control mRNA (lanes 1 – 7 ) and pre-mRNA (lanes 9 – 15 ).

Techniques Used: Incubation, Irradiation, Polyacrylamide Gel Electrophoresis, Fractionation, Modification, SDS Page

Splicing-dependent protection of 5′ exon intermediates from RNase cleavage. ( A ) Uniformly labeled AdML-GG pre-mRNA (lanes 1 – 15 ) or control 5′ exon RNA (lanes 16 – 25 ) was incubated under standard splicing conditions in HeLa cell nuclear extracts for the times indicated. Aliquots of these reactions were further incubated with the cDNA oligonucleotides indicated (lanes 7 – 15,17 – 25 ). Note that in splicing reactions, the RNase H cleavage products migrating below the free 5′ exon derive from both pre-mRNA and free 5′ exons; therefore, 5′ exon intermediate protection is best monitored in splicing reactions by the proportion of intact free 5′ exon remaining after digestion. ( B ) Similar RNase H analysis using control 5′ exon and spliced α-TM RNAs. ( C ) Complete RNase digestion of singly labeled AdML RNAs. ( Upper panel) Splicing time courses for AdML pre-mRNAs containing a single labeled phosphate at position −24 relative to the 5′ splice site (15% denaturing PAGE). Splicing substrates, intermediates, and products are indicated to the right. ( Lower panel) Separation of protected fragments by denaturing PAGE (20%) after RNase A + T 1 digestion. Closed arrows indicate fragments corresponding to the 5′ exon intermediate, whereas the open arrow denotes the fragment corresponding to spliced mRNA. ( D ) Same as C except using PIP.B+10 pre-mRNA containing a single labeled phosphate at position −24 relative to the 5′ splice site.
Figure Legend Snippet: Splicing-dependent protection of 5′ exon intermediates from RNase cleavage. ( A ) Uniformly labeled AdML-GG pre-mRNA (lanes 1 – 15 ) or control 5′ exon RNA (lanes 16 – 25 ) was incubated under standard splicing conditions in HeLa cell nuclear extracts for the times indicated. Aliquots of these reactions were further incubated with the cDNA oligonucleotides indicated (lanes 7 – 15,17 – 25 ). Note that in splicing reactions, the RNase H cleavage products migrating below the free 5′ exon derive from both pre-mRNA and free 5′ exons; therefore, 5′ exon intermediate protection is best monitored in splicing reactions by the proportion of intact free 5′ exon remaining after digestion. ( B ) Similar RNase H analysis using control 5′ exon and spliced α-TM RNAs. ( C ) Complete RNase digestion of singly labeled AdML RNAs. ( Upper panel) Splicing time courses for AdML pre-mRNAs containing a single labeled phosphate at position −24 relative to the 5′ splice site (15% denaturing PAGE). Splicing substrates, intermediates, and products are indicated to the right. ( Lower panel) Separation of protected fragments by denaturing PAGE (20%) after RNase A + T 1 digestion. Closed arrows indicate fragments corresponding to the 5′ exon intermediate, whereas the open arrow denotes the fragment corresponding to spliced mRNA. ( D ) Same as C except using PIP.B+10 pre-mRNA containing a single labeled phosphate at position −24 relative to the 5′ splice site.

Techniques Used: Labeling, Incubation, Polyacrylamide Gel Electrophoresis

52) Product Images from "Single-stranded RNA facilitates nucleocapsid: APOBEC3G complex formation"

Article Title: Single-stranded RNA facilitates nucleocapsid: APOBEC3G complex formation

Journal: RNA

doi: 10.1261/rna.964708

RNase A treatment of A3G blocks formation of an A3G:NC complex. A3G-Myc-His was purified from transfected 293T cells and either left untreated or incubated with RNase A, as indicated. Similarly, GST-NC was purified from bacteria and left untreated or incubated with RNase A. Treated or untreated protein samples were then mixed and incubated together, and any A3G:NC complexes collected using glutathione-agarose beads. The input A3G proteins are shown in the upper panel, lanes 1 and 2 , while the bound A3G proteins are shown in lanes 3–6 . The GST-NC protein used is shown in the lower panel. Proteins were visualized by Western analysis using a rabbit polyclonal anti-A3G antiserum ( upper panel) or an anti-GST polyclonal antibody ( lower panel).
Figure Legend Snippet: RNase A treatment of A3G blocks formation of an A3G:NC complex. A3G-Myc-His was purified from transfected 293T cells and either left untreated or incubated with RNase A, as indicated. Similarly, GST-NC was purified from bacteria and left untreated or incubated with RNase A. Treated or untreated protein samples were then mixed and incubated together, and any A3G:NC complexes collected using glutathione-agarose beads. The input A3G proteins are shown in the upper panel, lanes 1 and 2 , while the bound A3G proteins are shown in lanes 3–6 . The GST-NC protein used is shown in the lower panel. Proteins were visualized by Western analysis using a rabbit polyclonal anti-A3G antiserum ( upper panel) or an anti-GST polyclonal antibody ( lower panel).

Techniques Used: Purification, Transfection, Incubation, Western Blot

Formation of the A3G:NC complex is mediated by unstructured RNAs. ( A ) The purified A3G-Myc-His was untreated (lanes 3 , 4 ) or had been incubated with RNase A (all other lanes). Input A3G-Myc-His protein (5% of total) is shown in lanes 1 and 2 . A3G-Myc-His protein was incubated with purified GST (lane 3 ) or GST-NC (lanes 4–10 ). In lanes 6–10 , yeast RNA was added to the incubation: lane 6 , 1.25 μg; lane 7 , 2.5 μg; lane 8 , 5 μg; lane 9 , 10 μg; lane 10 , 20 μg. A3G:NC complexes were recovered using glutathione-agarose beads and bound A3G-Myc-His protein visualized by Western analysis using a rabbit polyclonal anti-A3G antiserum ( upper panel). Input GST or GST-NC protein was also analyzed by Western blot ( lower panel). ( B ) Similar to panel A , except that the added RNAs (10 μg in each case) represent yeast total RNA (lane 3 ); purified yeast tRNA (lane 4 ); total human cell RNA (lane 5 ), or total human RNA that had been subjected to one round (lane 6 ) or two rounds (lane 7 ) of purification on an oligo-dT column. This experiment used exclusively RNase A treated A3G-Myc-His protein. ( C ) Ethidium bromide-stained agarose gel visualizing 5 μg of each of the RNA samples used in panel B . As may be seen, the yeast RNA sample (Ambion) in lane 1 is degraded to the point where it is smaller than the 70–80-nt-long yeast tRNAs shown in lane 2 . Lanes 3–5 reveal the removal of human rRNA as the total human RNA sample (lane 3 ) was subjected to one (lane 4 ) or two (lane 5 ) rounds of oligo-dT purification. ( D ) Similar to panels A and B , except that this experiment also analyzed in vitro transcribed human Y4 RNA and 7SL RNA.
Figure Legend Snippet: Formation of the A3G:NC complex is mediated by unstructured RNAs. ( A ) The purified A3G-Myc-His was untreated (lanes 3 , 4 ) or had been incubated with RNase A (all other lanes). Input A3G-Myc-His protein (5% of total) is shown in lanes 1 and 2 . A3G-Myc-His protein was incubated with purified GST (lane 3 ) or GST-NC (lanes 4–10 ). In lanes 6–10 , yeast RNA was added to the incubation: lane 6 , 1.25 μg; lane 7 , 2.5 μg; lane 8 , 5 μg; lane 9 , 10 μg; lane 10 , 20 μg. A3G:NC complexes were recovered using glutathione-agarose beads and bound A3G-Myc-His protein visualized by Western analysis using a rabbit polyclonal anti-A3G antiserum ( upper panel). Input GST or GST-NC protein was also analyzed by Western blot ( lower panel). ( B ) Similar to panel A , except that the added RNAs (10 μg in each case) represent yeast total RNA (lane 3 ); purified yeast tRNA (lane 4 ); total human cell RNA (lane 5 ), or total human RNA that had been subjected to one round (lane 6 ) or two rounds (lane 7 ) of purification on an oligo-dT column. This experiment used exclusively RNase A treated A3G-Myc-His protein. ( C ) Ethidium bromide-stained agarose gel visualizing 5 μg of each of the RNA samples used in panel B . As may be seen, the yeast RNA sample (Ambion) in lane 1 is degraded to the point where it is smaller than the 70–80-nt-long yeast tRNAs shown in lane 2 . Lanes 3–5 reveal the removal of human rRNA as the total human RNA sample (lane 3 ) was subjected to one (lane 4 ) or two (lane 5 ) rounds of oligo-dT purification. ( D ) Similar to panels A and B , except that this experiment also analyzed in vitro transcribed human Y4 RNA and 7SL RNA.

Techniques Used: Purification, Incubation, Western Blot, Staining, Agarose Gel Electrophoresis, In Vitro

53) Product Images from "Polyarginine as a multifunctional fusion tag"

Article Title: Polyarginine as a multifunctional fusion tag

Journal: Protein Science : A Publication of the Protein Society

doi: 10.1110/ps.051393805

Effect of an R 9 tag on the purification of a protein by cation-exchange chromatography. ( A ) RNase A-R 9 was purified by cation-exchange chromatography before (−CPB) and after (+CPB) the addition of carboxypeptidase B. ( B ) SDS-PAGE gel of RNase A-R 9 before (−CPB) and after (+CPB) the addition of carboxypeptidase B. Purified RNase A is a standard.
Figure Legend Snippet: Effect of an R 9 tag on the purification of a protein by cation-exchange chromatography. ( A ) RNase A-R 9 was purified by cation-exchange chromatography before (−CPB) and after (+CPB) the addition of carboxypeptidase B. ( B ) SDS-PAGE gel of RNase A-R 9 before (−CPB) and after (+CPB) the addition of carboxypeptidase B. Purified RNase A is a standard.

Techniques Used: Purification, Chromatography, SDS Page

Effect of an R 9 tag on the adsorption of a protein to a glass slide and silica resin. ( A ) Fluorescent images of fluorescein-labeled RNase A-R 9 and RNase A (10–0.01 μM) adsorbed on to a glass slide. ( B ) Ribonucleolytic activity in a solution containing silica resin with adsorbed RNase A-R 9 or RNase A, and in the supernatant upon removal of the silica resin with adsorbed RNase A-R 9 .
Figure Legend Snippet: Effect of an R 9 tag on the adsorption of a protein to a glass slide and silica resin. ( A ) Fluorescent images of fluorescein-labeled RNase A-R 9 and RNase A (10–0.01 μM) adsorbed on to a glass slide. ( B ) Ribonucleolytic activity in a solution containing silica resin with adsorbed RNase A-R 9 or RNase A, and in the supernatant upon removal of the silica resin with adsorbed RNase A-R 9 .

Techniques Used: Adsorption, Labeling, Activity Assay

Effect of an R 9 tag on the uptake of a protein by living mammalian cells. CHO-K1 cells were incubated with fluorescein-labeled RNase A-R 9 (10 μM, A ) or fluorescein-labeled RNase A (10 μM, B ) for 15 min at 37°C before visualization by fluorescence microscopy. Scale bar: 10 μm.
Figure Legend Snippet: Effect of an R 9 tag on the uptake of a protein by living mammalian cells. CHO-K1 cells were incubated with fluorescein-labeled RNase A-R 9 (10 μM, A ) or fluorescein-labeled RNase A (10 μM, B ) for 15 min at 37°C before visualization by fluorescence microscopy. Scale bar: 10 μm.

Techniques Used: Incubation, Labeling, Fluorescence, Microscopy

54) Product Images from "Refolding and simultaneous purification by three-phase partitioning of recombinant proteins from inclusion bodies"

Article Title: Refolding and simultaneous purification by three-phase partitioning of recombinant proteins from inclusion bodies

Journal: Protein Science : A Publication of the Protein Society

doi: 10.1110/ps.036939.108

The binding isotherm of RNase A and 3′-CMP. RNase A (55 μM) in 0.2 M sodium acetate buffer, pH 6.0 containing 0.2 M NaCl, was titrated with small injections of 3′-CMP solution in the same buffer. The enthalpy data were fitted to a single site binding model. ( A ) Binding isotherm for refolded RNase A; ( B ) binding isotherm for Sigma RNase A.
Figure Legend Snippet: The binding isotherm of RNase A and 3′-CMP. RNase A (55 μM) in 0.2 M sodium acetate buffer, pH 6.0 containing 0.2 M NaCl, was titrated with small injections of 3′-CMP solution in the same buffer. The enthalpy data were fitted to a single site binding model. ( A ) Binding isotherm for refolded RNase A; ( B ) binding isotherm for Sigma RNase A.

Techniques Used: Binding Assay

Spectroscopic characterization of refolded RNase A. ( A ) Fluorescence emission spectra of RNase A. The samples at a concentration of 100 μg/mL −1 (∼7 μM) were excited at 278 nm and the emission spectra from 290 to 400 nm were recorded, using excitation and emission slit widths of 2 nm and 5 nm, respectively. Fluorescence spectra of unfolded RNase A (3), refolded RNase A (2), Sigma RNase A (1) are shown after correction for buffer contribution. ( B ) The far-UV CD spectra of refolded RNase A (○) and Sigma RNase A (△) for 0.5 mg/mL −1 protein recorded in a 1-mm path length cuvette from 200 to 250 nm. ( C ) The near-UV CD spectra of the two samples recorded for 0.5 mg/mL −1 protein in a 1-cm path length cuvette from 250 to 350 nm. The symbols are the same as in B .
Figure Legend Snippet: Spectroscopic characterization of refolded RNase A. ( A ) Fluorescence emission spectra of RNase A. The samples at a concentration of 100 μg/mL −1 (∼7 μM) were excited at 278 nm and the emission spectra from 290 to 400 nm were recorded, using excitation and emission slit widths of 2 nm and 5 nm, respectively. Fluorescence spectra of unfolded RNase A (3), refolded RNase A (2), Sigma RNase A (1) are shown after correction for buffer contribution. ( B ) The far-UV CD spectra of refolded RNase A (○) and Sigma RNase A (△) for 0.5 mg/mL −1 protein recorded in a 1-mm path length cuvette from 200 to 250 nm. ( C ) The near-UV CD spectra of the two samples recorded for 0.5 mg/mL −1 protein in a 1-cm path length cuvette from 250 to 350 nm. The symbols are the same as in B .

Techniques Used: Fluorescence, Concentration Assay

TPP purification monitored by SDS-PAGE. ( A ) Fifteen percent SDS-PAGE analysis of RNase A inclusion bodies at different steps of TPP; (lane 1 ) unwashed inclusion bodies; (lane 2 ) washed inclusion bodies with 50 mM PBS/pH 7.4/1 mM EDTA containing 2 M urea; (lane 3 ) commercial RNase A (Sigma Chemical Co.); (lane 4 ) refolded RNase A obtained from interfacial precipitate of second TPP; (lane 5 ) interfacial precipitate of first TPP; (lane 6 ) aqueous phase of second TPP. ( B ) Fifteen percent SDS-PAGE analysis of inclusion bodies of CcdB mutants subjected to TPP; (lane 1 ) unwashed inclusion bodies of CcdB-F17P; (lane 2 ) washed inclusion bodies of CcdB-F17P with 50 mM PBS/pH 7.4/0.5% Triton X-100; (lane 3 ) interfacial precipitate of first TPP; (lane 4 ) aqueous phase of second TPP; (lane 5 ) refolded and purified CcdB-F17P by TPP; (lane 6 ) molecular weight marker; (lane 7 ) unwashed inclusion bodies of CcdB-M97K; (lane 8 ) washed inclusion bodies of CcdB-M97K; (lane 9 ) interfacial precipitate of first TPP; (lane 10 ) aqueous phase of second TPP; (lane 11 ) refolded and purified CcdB-M97K by TPP. ( C ) Fifteen percent SDS-PAGE analysis of inclusion bodies of CD4D12 subjected to TPP CD4D12; (lane 1 ) molecular weight marker; (lane 2 ) unwashed inclusion bodies of CD4D12; (lane 3 ) washed inclusion bodies of CD4D12 with 50 mM PBS/pH 7.4/0.5% Triton X-100; (lane 4 ) interfacial precipitate of first TPP; (lane 5 ) aqueous phase of second TPP; (lane 6 ) refolded and purified CD4D12 by TPP; (lane 7 ) refolded and purified CD4D12 after subjected to the 50-kDa polyethersulfone membrane (PALL Lifesciences) once; (lane 8 ) refolded and purified CD4D12 after subjected to the 50-kDa polyethersulfone membrane (PALL Lifesciences) twice; (lane 9 ) refolded and purified CD4D12 in the absence of DTT.
Figure Legend Snippet: TPP purification monitored by SDS-PAGE. ( A ) Fifteen percent SDS-PAGE analysis of RNase A inclusion bodies at different steps of TPP; (lane 1 ) unwashed inclusion bodies; (lane 2 ) washed inclusion bodies with 50 mM PBS/pH 7.4/1 mM EDTA containing 2 M urea; (lane 3 ) commercial RNase A (Sigma Chemical Co.); (lane 4 ) refolded RNase A obtained from interfacial precipitate of second TPP; (lane 5 ) interfacial precipitate of first TPP; (lane 6 ) aqueous phase of second TPP. ( B ) Fifteen percent SDS-PAGE analysis of inclusion bodies of CcdB mutants subjected to TPP; (lane 1 ) unwashed inclusion bodies of CcdB-F17P; (lane 2 ) washed inclusion bodies of CcdB-F17P with 50 mM PBS/pH 7.4/0.5% Triton X-100; (lane 3 ) interfacial precipitate of first TPP; (lane 4 ) aqueous phase of second TPP; (lane 5 ) refolded and purified CcdB-F17P by TPP; (lane 6 ) molecular weight marker; (lane 7 ) unwashed inclusion bodies of CcdB-M97K; (lane 8 ) washed inclusion bodies of CcdB-M97K; (lane 9 ) interfacial precipitate of first TPP; (lane 10 ) aqueous phase of second TPP; (lane 11 ) refolded and purified CcdB-M97K by TPP. ( C ) Fifteen percent SDS-PAGE analysis of inclusion bodies of CD4D12 subjected to TPP CD4D12; (lane 1 ) molecular weight marker; (lane 2 ) unwashed inclusion bodies of CD4D12; (lane 3 ) washed inclusion bodies of CD4D12 with 50 mM PBS/pH 7.4/0.5% Triton X-100; (lane 4 ) interfacial precipitate of first TPP; (lane 5 ) aqueous phase of second TPP; (lane 6 ) refolded and purified CD4D12 by TPP; (lane 7 ) refolded and purified CD4D12 after subjected to the 50-kDa polyethersulfone membrane (PALL Lifesciences) once; (lane 8 ) refolded and purified CD4D12 after subjected to the 50-kDa polyethersulfone membrane (PALL Lifesciences) twice; (lane 9 ) refolded and purified CD4D12 in the absence of DTT.

Techniques Used: Purification, SDS Page, Molecular Weight, Marker

55) Product Images from "The MRB1 complex functions in kinetoplastid RNA processing"

Article Title: The MRB1 complex functions in kinetoplastid RNA processing

Journal: RNA

doi: 10.1261/rna.1353209

The MRB1 complex binds to kDNA transcripts. Western analysis of mt vesicles isolated from wt PF cells, lysed in the presence (+) or absence (−) of 0.1 mg/mL RNase A, and fractionated on 10%–30% glycerol gradients. Every other 30 μL
Figure Legend Snippet: The MRB1 complex binds to kDNA transcripts. Western analysis of mt vesicles isolated from wt PF cells, lysed in the presence (+) or absence (−) of 0.1 mg/mL RNase A, and fractionated on 10%–30% glycerol gradients. Every other 30 μL

Techniques Used: Western Blot, Isolation

56) Product Images from "Refolding and simultaneous purification by three-phase partitioning of recombinant proteins from inclusion bodies"

Article Title: Refolding and simultaneous purification by three-phase partitioning of recombinant proteins from inclusion bodies

Journal: Protein Science : A Publication of the Protein Society

doi: 10.1110/ps.036939.108

The binding isotherm of RNase A and 3′-CMP. RNase A (55 μM) in 0.2 M sodium acetate buffer, pH 6.0 containing 0.2 M NaCl, was titrated with small injections of 3′-CMP solution in the same buffer. The enthalpy data were fitted to a single site binding model. ( A ) Binding isotherm for refolded RNase A; ( B ) binding isotherm for Sigma RNase A.
Figure Legend Snippet: The binding isotherm of RNase A and 3′-CMP. RNase A (55 μM) in 0.2 M sodium acetate buffer, pH 6.0 containing 0.2 M NaCl, was titrated with small injections of 3′-CMP solution in the same buffer. The enthalpy data were fitted to a single site binding model. ( A ) Binding isotherm for refolded RNase A; ( B ) binding isotherm for Sigma RNase A.

Techniques Used: Binding Assay

Spectroscopic characterization of refolded RNase A. ( A ) Fluorescence emission spectra of RNase A. The samples at a concentration of 100 μg/mL −1 (∼7 μM) were excited at 278 nm and the emission spectra from 290 to 400 nm were recorded, using excitation and emission slit widths of 2 nm and 5 nm, respectively. Fluorescence spectra of unfolded RNase A (3), refolded RNase A (2), Sigma RNase A (1) are shown after correction for buffer contribution. ( B ) The far-UV CD spectra of refolded RNase A (○) and Sigma RNase A (△) for 0.5 mg/mL −1 protein recorded in a 1-mm path length cuvette from 200 to 250 nm. ( C ) The near-UV CD spectra of the two samples recorded for 0.5 mg/mL −1 protein in a 1-cm path length cuvette from 250 to 350 nm. The symbols are the same as in B .
Figure Legend Snippet: Spectroscopic characterization of refolded RNase A. ( A ) Fluorescence emission spectra of RNase A. The samples at a concentration of 100 μg/mL −1 (∼7 μM) were excited at 278 nm and the emission spectra from 290 to 400 nm were recorded, using excitation and emission slit widths of 2 nm and 5 nm, respectively. Fluorescence spectra of unfolded RNase A (3), refolded RNase A (2), Sigma RNase A (1) are shown after correction for buffer contribution. ( B ) The far-UV CD spectra of refolded RNase A (○) and Sigma RNase A (△) for 0.5 mg/mL −1 protein recorded in a 1-mm path length cuvette from 200 to 250 nm. ( C ) The near-UV CD spectra of the two samples recorded for 0.5 mg/mL −1 protein in a 1-cm path length cuvette from 250 to 350 nm. The symbols are the same as in B .

Techniques Used: Fluorescence, Concentration Assay

TPP purification monitored by SDS-PAGE. ( A ) Fifteen percent SDS-PAGE analysis of RNase A inclusion bodies at different steps of TPP; (lane 1 ) unwashed inclusion bodies; (lane 2 ) washed inclusion bodies with 50 mM PBS/pH 7.4/1 mM EDTA containing 2 M urea; (lane 3 ) commercial RNase A (Sigma Chemical Co.); (lane 4 ) refolded RNase A obtained from interfacial precipitate of second TPP; (lane 5 ) interfacial precipitate of first TPP; (lane 6 ) aqueous phase of second TPP. ( B ) Fifteen percent SDS-PAGE analysis of inclusion bodies of CcdB mutants subjected to TPP; (lane 1 ) unwashed inclusion bodies of CcdB-F17P; (lane 2 ) washed inclusion bodies of CcdB-F17P with 50 mM PBS/pH 7.4/0.5% Triton X-100; (lane 3 ) interfacial precipitate of first TPP; (lane 4 ) aqueous phase of second TPP; (lane 5 ) refolded and purified CcdB-F17P by TPP; (lane 6 ) molecular weight marker; (lane 7 ) unwashed inclusion bodies of CcdB-M97K; (lane 8 ) washed inclusion bodies of CcdB-M97K; (lane 9 ) interfacial precipitate of first TPP; (lane 10 ) aqueous phase of second TPP; (lane 11 ) refolded and purified CcdB-M97K by TPP. ( C ) Fifteen percent SDS-PAGE analysis of inclusion bodies of CD4D12 subjected to TPP CD4D12; (lane 1 ) molecular weight marker; (lane 2 ) unwashed inclusion bodies of CD4D12; (lane 3 ) washed inclusion bodies of CD4D12 with 50 mM PBS/pH 7.4/0.5% Triton X-100; (lane 4 ) interfacial precipitate of first TPP; (lane 5 ) aqueous phase of second TPP; (lane 6 ) refolded and purified CD4D12 by TPP; (lane 7 ) refolded and purified CD4D12 after subjected to the 50-kDa polyethersulfone membrane (PALL Lifesciences) once; (lane 8 ) refolded and purified CD4D12 after subjected to the 50-kDa polyethersulfone membrane (PALL Lifesciences) twice; (lane 9 ) refolded and purified CD4D12 in the absence of DTT.
Figure Legend Snippet: TPP purification monitored by SDS-PAGE. ( A ) Fifteen percent SDS-PAGE analysis of RNase A inclusion bodies at different steps of TPP; (lane 1 ) unwashed inclusion bodies; (lane 2 ) washed inclusion bodies with 50 mM PBS/pH 7.4/1 mM EDTA containing 2 M urea; (lane 3 ) commercial RNase A (Sigma Chemical Co.); (lane 4 ) refolded RNase A obtained from interfacial precipitate of second TPP; (lane 5 ) interfacial precipitate of first TPP; (lane 6 ) aqueous phase of second TPP. ( B ) Fifteen percent SDS-PAGE analysis of inclusion bodies of CcdB mutants subjected to TPP; (lane 1 ) unwashed inclusion bodies of CcdB-F17P; (lane 2 ) washed inclusion bodies of CcdB-F17P with 50 mM PBS/pH 7.4/0.5% Triton X-100; (lane 3 ) interfacial precipitate of first TPP; (lane 4 ) aqueous phase of second TPP; (lane 5 ) refolded and purified CcdB-F17P by TPP; (lane 6 ) molecular weight marker; (lane 7 ) unwashed inclusion bodies of CcdB-M97K; (lane 8 ) washed inclusion bodies of CcdB-M97K; (lane 9 ) interfacial precipitate of first TPP; (lane 10 ) aqueous phase of second TPP; (lane 11 ) refolded and purified CcdB-M97K by TPP. ( C ) Fifteen percent SDS-PAGE analysis of inclusion bodies of CD4D12 subjected to TPP CD4D12; (lane 1 ) molecular weight marker; (lane 2 ) unwashed inclusion bodies of CD4D12; (lane 3 ) washed inclusion bodies of CD4D12 with 50 mM PBS/pH 7.4/0.5% Triton X-100; (lane 4 ) interfacial precipitate of first TPP; (lane 5 ) aqueous phase of second TPP; (lane 6 ) refolded and purified CD4D12 by TPP; (lane 7 ) refolded and purified CD4D12 after subjected to the 50-kDa polyethersulfone membrane (PALL Lifesciences) once; (lane 8 ) refolded and purified CD4D12 after subjected to the 50-kDa polyethersulfone membrane (PALL Lifesciences) twice; (lane 9 ) refolded and purified CD4D12 in the absence of DTT.

Techniques Used: Purification, SDS Page, Molecular Weight, Marker

57) Product Images from "Refolding and simultaneous purification by three-phase partitioning of recombinant proteins from inclusion bodies"

Article Title: Refolding and simultaneous purification by three-phase partitioning of recombinant proteins from inclusion bodies

Journal: Protein Science : A Publication of the Protein Society

doi: 10.1110/ps.036939.108

The binding isotherm of RNase A and 3′-CMP. RNase A (55 μM) in 0.2 M sodium acetate buffer, pH 6.0 containing 0.2 M NaCl, was titrated with small injections of 3′-CMP solution in the same buffer. The enthalpy data were fitted to a single site binding model. ( A ) Binding isotherm for refolded RNase A; ( B ) binding isotherm for Sigma RNase A.
Figure Legend Snippet: The binding isotherm of RNase A and 3′-CMP. RNase A (55 μM) in 0.2 M sodium acetate buffer, pH 6.0 containing 0.2 M NaCl, was titrated with small injections of 3′-CMP solution in the same buffer. The enthalpy data were fitted to a single site binding model. ( A ) Binding isotherm for refolded RNase A; ( B ) binding isotherm for Sigma RNase A.

Techniques Used: Binding Assay

Spectroscopic characterization of refolded RNase A. ( A ) Fluorescence emission spectra of RNase A. The samples at a concentration of 100 μg/mL −1 (∼7 μM) were excited at 278 nm and the emission spectra from 290 to 400 nm were recorded, using excitation and emission slit widths of 2 nm and 5 nm, respectively. Fluorescence spectra of unfolded RNase A (3), refolded RNase A (2), Sigma RNase A (1) are shown after correction for buffer contribution. ( B ) The far-UV CD spectra of refolded RNase A (○) and Sigma RNase A (△) for 0.5 mg/mL −1 protein recorded in a 1-mm path length cuvette from 200 to 250 nm. ( C ) The near-UV CD spectra of the two samples recorded for 0.5 mg/mL −1 protein in a 1-cm path length cuvette from 250 to 350 nm. The symbols are the same as in B .
Figure Legend Snippet: Spectroscopic characterization of refolded RNase A. ( A ) Fluorescence emission spectra of RNase A. The samples at a concentration of 100 μg/mL −1 (∼7 μM) were excited at 278 nm and the emission spectra from 290 to 400 nm were recorded, using excitation and emission slit widths of 2 nm and 5 nm, respectively. Fluorescence spectra of unfolded RNase A (3), refolded RNase A (2), Sigma RNase A (1) are shown after correction for buffer contribution. ( B ) The far-UV CD spectra of refolded RNase A (○) and Sigma RNase A (△) for 0.5 mg/mL −1 protein recorded in a 1-mm path length cuvette from 200 to 250 nm. ( C ) The near-UV CD spectra of the two samples recorded for 0.5 mg/mL −1 protein in a 1-cm path length cuvette from 250 to 350 nm. The symbols are the same as in B .

Techniques Used: Fluorescence, Concentration Assay

TPP purification monitored by SDS-PAGE. ( A ) Fifteen percent SDS-PAGE analysis of RNase A inclusion bodies at different steps of TPP; (lane 1 ) unwashed inclusion bodies; (lane 2 ) washed inclusion bodies with 50 mM PBS/pH 7.4/1 mM EDTA containing 2 M urea; (lane 3 ) commercial RNase A (Sigma Chemical Co.); (lane 4 ) refolded RNase A obtained from interfacial precipitate of second TPP; (lane 5 ) interfacial precipitate of first TPP; (lane 6 ) aqueous phase of second TPP. ( B ) Fifteen percent SDS-PAGE analysis of inclusion bodies of CcdB mutants subjected to TPP; (lane 1 ) unwashed inclusion bodies of CcdB-F17P; (lane 2 ) washed inclusion bodies of CcdB-F17P with 50 mM PBS/pH 7.4/0.5% Triton X-100; (lane 3 ) interfacial precipitate of first TPP; (lane 4 ) aqueous phase of second TPP; (lane 5 ) refolded and purified CcdB-F17P by TPP; (lane 6 ) molecular weight marker; (lane 7 ) unwashed inclusion bodies of CcdB-M97K; (lane 8 ) washed inclusion bodies of CcdB-M97K; (lane 9 ) interfacial precipitate of first TPP; (lane 10 ) aqueous phase of second TPP; (lane 11 ) refolded and purified CcdB-M97K by TPP. ( C ) Fifteen percent SDS-PAGE analysis of inclusion bodies of CD4D12 subjected to TPP CD4D12; (lane 1 ) molecular weight marker; (lane 2 ) unwashed inclusion bodies of CD4D12; (lane 3 ) washed inclusion bodies of CD4D12 with 50 mM PBS/pH 7.4/0.5% Triton X-100; (lane 4 ) interfacial precipitate of first TPP; (lane 5 ) aqueous phase of second TPP; (lane 6 ) refolded and purified CD4D12 by TPP; (lane 7 ) refolded and purified CD4D12 after subjected to the 50-kDa polyethersulfone membrane (PALL Lifesciences) once; (lane 8 ) refolded and purified CD4D12 after subjected to the 50-kDa polyethersulfone membrane (PALL Lifesciences) twice; (lane 9 ) refolded and purified CD4D12 in the absence of DTT.
Figure Legend Snippet: TPP purification monitored by SDS-PAGE. ( A ) Fifteen percent SDS-PAGE analysis of RNase A inclusion bodies at different steps of TPP; (lane 1 ) unwashed inclusion bodies; (lane 2 ) washed inclusion bodies with 50 mM PBS/pH 7.4/1 mM EDTA containing 2 M urea; (lane 3 ) commercial RNase A (Sigma Chemical Co.); (lane 4 ) refolded RNase A obtained from interfacial precipitate of second TPP; (lane 5 ) interfacial precipitate of first TPP; (lane 6 ) aqueous phase of second TPP. ( B ) Fifteen percent SDS-PAGE analysis of inclusion bodies of CcdB mutants subjected to TPP; (lane 1 ) unwashed inclusion bodies of CcdB-F17P; (lane 2 ) washed inclusion bodies of CcdB-F17P with 50 mM PBS/pH 7.4/0.5% Triton X-100; (lane 3 ) interfacial precipitate of first TPP; (lane 4 ) aqueous phase of second TPP; (lane 5 ) refolded and purified CcdB-F17P by TPP; (lane 6 ) molecular weight marker; (lane 7 ) unwashed inclusion bodies of CcdB-M97K; (lane 8 ) washed inclusion bodies of CcdB-M97K; (lane 9 ) interfacial precipitate of first TPP; (lane 10 ) aqueous phase of second TPP; (lane 11 ) refolded and purified CcdB-M97K by TPP. ( C ) Fifteen percent SDS-PAGE analysis of inclusion bodies of CD4D12 subjected to TPP CD4D12; (lane 1 ) molecular weight marker; (lane 2 ) unwashed inclusion bodies of CD4D12; (lane 3 ) washed inclusion bodies of CD4D12 with 50 mM PBS/pH 7.4/0.5% Triton X-100; (lane 4 ) interfacial precipitate of first TPP; (lane 5 ) aqueous phase of second TPP; (lane 6 ) refolded and purified CD4D12 by TPP; (lane 7 ) refolded and purified CD4D12 after subjected to the 50-kDa polyethersulfone membrane (PALL Lifesciences) once; (lane 8 ) refolded and purified CD4D12 after subjected to the 50-kDa polyethersulfone membrane (PALL Lifesciences) twice; (lane 9 ) refolded and purified CD4D12 in the absence of DTT.

Techniques Used: Purification, SDS Page, Molecular Weight, Marker

58) Product Images from "Identification of Interactions in the NMD Complex Using Proximity-Dependent Biotinylation (BioID)"

Article Title: Identification of Interactions in the NMD Complex Using Proximity-Dependent Biotinylation (BioID)

Journal: PLoS ONE

doi: 10.1371/journal.pone.0150239

UPF2 transiently interacts with translation factors. (A) Western blots of anti-HA co-immunoprecipitation experiment from 293T cells overexpressing H16-UPF2 R . Protein hyperphosphorylation, dephosphorylation and RNase A treatment conditions were as in Fig 7 . (B) Western blot of co-immunoprecipitation of endogenous EIF4A2 from 293T cells with an antibody against the n -terminus of EIF4A2 (lanes 3,4, 6,7) either with (lanes 4 and 7) or without RNase A treatment (lanes 3 and 6). Whole IgG was used as a control (lanes 2 and 5). Input, unbound (5x10 5 cell equivalents each) and immunoprecipitated material (equivalent to 1x10 7 cells) was used in (A) and (B). White and black triangles denote the position of the overexpressed and endogenous proteins, respectively. (C-E) Proximity ligation assays (PLA) probing for the effect translation inhibition on the pairwise co-localization of UPF2 and EIF4A2 (C), UPF2 and ribosomal protein RPS2 (D), and UPF2 and SMG1 (E). HeLa Tet-Off TCRβ ter68 cells (clone 2.2) were grown and where indicated treated with puromycin (Puro), cycloheximide (CHX) or arsentite. The antibody pairs for detection are depicted above the images. Omission of one or both primary antibodies controlled for the specificity of the PLA signal.
Figure Legend Snippet: UPF2 transiently interacts with translation factors. (A) Western blots of anti-HA co-immunoprecipitation experiment from 293T cells overexpressing H16-UPF2 R . Protein hyperphosphorylation, dephosphorylation and RNase A treatment conditions were as in Fig 7 . (B) Western blot of co-immunoprecipitation of endogenous EIF4A2 from 293T cells with an antibody against the n -terminus of EIF4A2 (lanes 3,4, 6,7) either with (lanes 4 and 7) or without RNase A treatment (lanes 3 and 6). Whole IgG was used as a control (lanes 2 and 5). Input, unbound (5x10 5 cell equivalents each) and immunoprecipitated material (equivalent to 1x10 7 cells) was used in (A) and (B). White and black triangles denote the position of the overexpressed and endogenous proteins, respectively. (C-E) Proximity ligation assays (PLA) probing for the effect translation inhibition on the pairwise co-localization of UPF2 and EIF4A2 (C), UPF2 and ribosomal protein RPS2 (D), and UPF2 and SMG1 (E). HeLa Tet-Off TCRβ ter68 cells (clone 2.2) were grown and where indicated treated with puromycin (Puro), cycloheximide (CHX) or arsentite. The antibody pairs for detection are depicted above the images. Omission of one or both primary antibodies controlled for the specificity of the PLA signal.

Techniques Used: Western Blot, Immunoprecipitation, De-Phosphorylation Assay, Ligation, Proximity Ligation Assay, Inhibition

Interaction of the NMD factors UPF1 and SMG5 with mRNP components and decapping factors. Western blots were performed to detect proteins that co-immunoprecipitate with H16-UPF1 R (A) or H16-SMG5 R (B) overexpressed in 293T cells. To boost the phsphorylation state of proteins, cells were treated with okadaic acid for 3 hours before harvesting and phosphatase (PPase) inhibitors were added to the lysates (lanes 4, 10, 11, 17, 18; highlighted by green retangles). To dephosphorylate proteins, cell lysates were incubated with PPase λ prior to IP (lanes 3, 8, 9, 15, 16; red rectangles). Finally, the RNA dependence of the associated proteins was assessed by RNase A treatment of lysates (lanes 7, 9, 11, 14, 16, 18). Input, unbound (5x10 5 cell equivalents each), and immunoprecipitated material (equivalent to 1x10 7 cells) were separated on SDS-PAGE, transferred to nitrocellulose membranes and probed with antibodies against the indicated proteins. Exact postions of overexpressed and endogenous proteins are indicated by white and black triangles, respectively, L denotes signal originating from the antibody light chain and HB bleed-through signal from the HA-BirA protein. For better visibility, a higher intensity scan of the membrane piece is shown in (B) for the XRN1 IP samples.
Figure Legend Snippet: Interaction of the NMD factors UPF1 and SMG5 with mRNP components and decapping factors. Western blots were performed to detect proteins that co-immunoprecipitate with H16-UPF1 R (A) or H16-SMG5 R (B) overexpressed in 293T cells. To boost the phsphorylation state of proteins, cells were treated with okadaic acid for 3 hours before harvesting and phosphatase (PPase) inhibitors were added to the lysates (lanes 4, 10, 11, 17, 18; highlighted by green retangles). To dephosphorylate proteins, cell lysates were incubated with PPase λ prior to IP (lanes 3, 8, 9, 15, 16; red rectangles). Finally, the RNA dependence of the associated proteins was assessed by RNase A treatment of lysates (lanes 7, 9, 11, 14, 16, 18). Input, unbound (5x10 5 cell equivalents each), and immunoprecipitated material (equivalent to 1x10 7 cells) were separated on SDS-PAGE, transferred to nitrocellulose membranes and probed with antibodies against the indicated proteins. Exact postions of overexpressed and endogenous proteins are indicated by white and black triangles, respectively, L denotes signal originating from the antibody light chain and HB bleed-through signal from the HA-BirA protein. For better visibility, a higher intensity scan of the membrane piece is shown in (B) for the XRN1 IP samples.

Techniques Used: Western Blot, Incubation, Immunoprecipitation, SDS Page

59) Product Images from "The complete chemical structure of Saccharomyces cerevisiae rRNA: partial pseudouridylation of U2345 in 25S rRNA by snoRNA snR9"

Article Title: The complete chemical structure of Saccharomyces cerevisiae rRNA: partial pseudouridylation of U2345 in 25S rRNA by snoRNA snR9

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkw564

Deletion of snR9 prevents pseudouridylation of U2345. ( A ) Potential base-pairing interactions between snR9 and Sc 25S rRNA. The upper sequence is that of the snoRNA snR9 with its hairpin shown as a solid line. The positions of A33, G67 and G69, which were mutated in our study, are indicted by the arrows. The lower sequence is that of the 25S rRNA around Ψ2345. The lower arrow points to Ψ2345. ( B ) Extracted ion chromatogram (EIC) of the fragments containing 2345 ΨmA 2347 Ψp produced by the RNase A digestion of the 25S rRNA from wild-type (upper panel), ΔsnR9 (middle panel) and ΔsnR33 (lower panel) Sc strains. The strains were cultured in the U/C-5-D labelling medium at 30°C and purified 25S rRNA was digested with RNase A. Each digest (50 fmol) was subjected to LC-MS. The sequence and m/z value of ΨmAΨp are shown in the figure. The most intense signal in the ΔsnR33 spectrum was set to a relative intensity of 100%, and the peaks in the wild-type spectrum were scaled accordingly. The arrows in the left panels indicate the position of the MS signal for the Ψm2345-containing fragment, which was not detected in the ΔsnR9 chromatogram.
Figure Legend Snippet: Deletion of snR9 prevents pseudouridylation of U2345. ( A ) Potential base-pairing interactions between snR9 and Sc 25S rRNA. The upper sequence is that of the snoRNA snR9 with its hairpin shown as a solid line. The positions of A33, G67 and G69, which were mutated in our study, are indicted by the arrows. The lower sequence is that of the 25S rRNA around Ψ2345. The lower arrow points to Ψ2345. ( B ) Extracted ion chromatogram (EIC) of the fragments containing 2345 ΨmA 2347 Ψp produced by the RNase A digestion of the 25S rRNA from wild-type (upper panel), ΔsnR9 (middle panel) and ΔsnR33 (lower panel) Sc strains. The strains were cultured in the U/C-5-D labelling medium at 30°C and purified 25S rRNA was digested with RNase A. Each digest (50 fmol) was subjected to LC-MS. The sequence and m/z value of ΨmAΨp are shown in the figure. The most intense signal in the ΔsnR33 spectrum was set to a relative intensity of 100%, and the peaks in the wild-type spectrum were scaled accordingly. The arrows in the left panels indicate the position of the MS signal for the Ψm2345-containing fragment, which was not detected in the ΔsnR9 chromatogram.

Techniques Used: Sequencing, Produced, Cell Culture, Purification, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

Episomal expression of snR9 in ΔsnR9_2 restores U2345 pseudouridylation. EICs of fragments from the RNase A digestion of 25S rRNA containing U2345 are shown. 25S rRNA was purified from ΔsnR9_2 that had been transformed with pSEC, pSECR9-WT, pSECR9-A33G, pSECR9-G67A or pSECR9-G69A. Each strain was cultured at 30°C in U/C-5-D labelling medium. The extracted 25S rRNAs were individually digested with RNase A, and the resulting digests (50 fmol) were subjected to LC-MS. The sequence and m/z value of ΨmAΨp are indicated. A mass window of ±10 ppm was used the chromatograms. The most intense peak in the pSECR-WT spectrum was set to 100%. The MS signal of ΨmAΨp was detected only in the spectrum of ΔsnR9 that had been transformed with pSECR9-WT, which enabled expression of wild-type snR9.
Figure Legend Snippet: Episomal expression of snR9 in ΔsnR9_2 restores U2345 pseudouridylation. EICs of fragments from the RNase A digestion of 25S rRNA containing U2345 are shown. 25S rRNA was purified from ΔsnR9_2 that had been transformed with pSEC, pSECR9-WT, pSECR9-A33G, pSECR9-G67A or pSECR9-G69A. Each strain was cultured at 30°C in U/C-5-D labelling medium. The extracted 25S rRNAs were individually digested with RNase A, and the resulting digests (50 fmol) were subjected to LC-MS. The sequence and m/z value of ΨmAΨp are indicated. A mass window of ±10 ppm was used the chromatograms. The most intense peak in the pSECR-WT spectrum was set to 100%. The MS signal of ΨmAΨp was detected only in the spectrum of ΔsnR9 that had been transformed with pSECR9-WT, which enabled expression of wild-type snR9.

Techniques Used: Expressing, Purification, Transformation Assay, Cell Culture, Liquid Chromatography with Mass Spectroscopy, Sequencing, Mass Spectrometry

60) Product Images from "Human Pat1b Connects Deadenylation with mRNA Decapping and Controls the Assembly of Processing Bodies ▿Human Pat1b Connects Deadenylation with mRNA Decapping and Controls the Assembly of Processing Bodies ▿ †"

Article Title: Human Pat1b Connects Deadenylation with mRNA Decapping and Controls the Assembly of Processing Bodies ▿Human Pat1b Connects Deadenylation with mRNA Decapping and Controls the Assembly of Processing Bodies ▿ †

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00429-10

Pat1b interacts with P-body proteins. (A) HEK293 cells were transiently transfected with vector alone, HA-tagged Pat1b, or HA-tagged Pat1a. After 1 day, cytoplasmic lysates (input) were prepared for IP with anti-HA antibody. The HA-tagged proteins as well as endogenous Rck, Hedls, Xrn1, Lsm1, Lsm4, and eIF3B were detected by Western blotting. The sizes of the molecular weight markers (in thousands) are indicated on the right. (B) HEK293 cells transiently transfected with vector alone, HA-Pat1b, or HA-Pat1b-YFP were used for IP as described in the legend for panel A. Where indicated, RNase A was added to the lysates during IP. Lanes 10 and 11 show RNA extracted from the unbound fraction and stained with ethidium bromide. (C) HA-Pat1b was immunoprecipitated with HA antibody or without antibody as a control for unspecific precipitation. (D) HA-Pat1b was immunoprecipitated and subjected to increasing NaCl concentrations prior to elution of the protein complexes. *, immunoglobulin heavy chain. (E) Endogenous Xrn1, Hedls, Rck, Lsm1, and 14-3-3 were immunoprecipitated from the cytoplasmic lysate of HEK293 cells transiently transfected with HA-Pat1b. The Western blot for Rck is not shown due to an overlapping signal from the immunoglobulin heavy chain.
Figure Legend Snippet: Pat1b interacts with P-body proteins. (A) HEK293 cells were transiently transfected with vector alone, HA-tagged Pat1b, or HA-tagged Pat1a. After 1 day, cytoplasmic lysates (input) were prepared for IP with anti-HA antibody. The HA-tagged proteins as well as endogenous Rck, Hedls, Xrn1, Lsm1, Lsm4, and eIF3B were detected by Western blotting. The sizes of the molecular weight markers (in thousands) are indicated on the right. (B) HEK293 cells transiently transfected with vector alone, HA-Pat1b, or HA-Pat1b-YFP were used for IP as described in the legend for panel A. Where indicated, RNase A was added to the lysates during IP. Lanes 10 and 11 show RNA extracted from the unbound fraction and stained with ethidium bromide. (C) HA-Pat1b was immunoprecipitated with HA antibody or without antibody as a control for unspecific precipitation. (D) HA-Pat1b was immunoprecipitated and subjected to increasing NaCl concentrations prior to elution of the protein complexes. *, immunoglobulin heavy chain. (E) Endogenous Xrn1, Hedls, Rck, Lsm1, and 14-3-3 were immunoprecipitated from the cytoplasmic lysate of HEK293 cells transiently transfected with HA-Pat1b. The Western blot for Rck is not shown due to an overlapping signal from the immunoglobulin heavy chain.

Techniques Used: Transfection, Plasmid Preparation, Western Blot, Molecular Weight, Staining, Immunoprecipitation

Pat1b interacts with the Ccr4-Caf1-Not complex. (A) HEK293 cells were transiently transfected with YFP or YFP-Pat1b together with Flag-Not1. GFP-binder was used for IP, and Western blot analysis was carried out with anti-GFP and anti-Flag antibody. The sizes of the molecular weight markers (in thousands) are indicated on the right. Where indicated, RNase A was added during IP. (B) IP was carried out as described in the legend for panel A using YFP or YFP-Pat1b together with myc-Ccr4; anti-myc was used for Western blot analysis. (C) IP was carried out as described in the legend for panel A using YFP or YFP-Pat1b together with HA-Caf1a or HA-Caf1b; anti-HA was used for Western blot analysis. On the right side, RNA was extracted from unbound fractions and stained with ethidium bromide. (D) HEK293 cells were transiently transfected with YFP, YFP-Pat1b, YFP-A, YFP-N, YFP-AN, or YFP-HC and processed for IP with GFP-binder. The YFP-tagged proteins and endogenous Caf1a were detected by Western blotting. (E) HEK293 cells were transiently cotransfected with YFP-Pat1b together with either Flag-Not1, myc-Ccr4, HA-Caf1a, or HA-Caf1b. IPs were carried out with GFP-binder and subjected to increasing NaCl concentrations prior to elution. (F) HEK293 cells were transiently transfected with Flag-Dcp2 together with either GFP alone, GFP-Caf1a alone, or GFP-Caf1a together with HA-Pat1b. Cytoplasmic lysates were processed for IP with GFP-binder and then subjected to Western blot analysis. In the IP samples, the Flag antibody cross-reacted with GFP-Caf1a.
Figure Legend Snippet: Pat1b interacts with the Ccr4-Caf1-Not complex. (A) HEK293 cells were transiently transfected with YFP or YFP-Pat1b together with Flag-Not1. GFP-binder was used for IP, and Western blot analysis was carried out with anti-GFP and anti-Flag antibody. The sizes of the molecular weight markers (in thousands) are indicated on the right. Where indicated, RNase A was added during IP. (B) IP was carried out as described in the legend for panel A using YFP or YFP-Pat1b together with myc-Ccr4; anti-myc was used for Western blot analysis. (C) IP was carried out as described in the legend for panel A using YFP or YFP-Pat1b together with HA-Caf1a or HA-Caf1b; anti-HA was used for Western blot analysis. On the right side, RNA was extracted from unbound fractions and stained with ethidium bromide. (D) HEK293 cells were transiently transfected with YFP, YFP-Pat1b, YFP-A, YFP-N, YFP-AN, or YFP-HC and processed for IP with GFP-binder. The YFP-tagged proteins and endogenous Caf1a were detected by Western blotting. (E) HEK293 cells were transiently cotransfected with YFP-Pat1b together with either Flag-Not1, myc-Ccr4, HA-Caf1a, or HA-Caf1b. IPs were carried out with GFP-binder and subjected to increasing NaCl concentrations prior to elution. (F) HEK293 cells were transiently transfected with Flag-Dcp2 together with either GFP alone, GFP-Caf1a alone, or GFP-Caf1a together with HA-Pat1b. Cytoplasmic lysates were processed for IP with GFP-binder and then subjected to Western blot analysis. In the IP samples, the Flag antibody cross-reacted with GFP-Caf1a.

Techniques Used: Transfection, Western Blot, Molecular Weight, Staining

61) Product Images from "Refolding and simultaneous purification by three-phase partitioning of recombinant proteins from inclusion bodies"

Article Title: Refolding and simultaneous purification by three-phase partitioning of recombinant proteins from inclusion bodies

Journal: Protein Science : A Publication of the Protein Society

doi: 10.1110/ps.036939.108

The binding isotherm of RNase A and 3′-CMP. RNase A (55 μM) in 0.2 M sodium acetate buffer, pH 6.0 containing 0.2 M NaCl, was titrated with small injections of 3′-CMP solution in the same buffer. The enthalpy data were fitted to a single site binding model. ( A ) Binding isotherm for refolded RNase A; ( B ) binding isotherm for Sigma RNase A.
Figure Legend Snippet: The binding isotherm of RNase A and 3′-CMP. RNase A (55 μM) in 0.2 M sodium acetate buffer, pH 6.0 containing 0.2 M NaCl, was titrated with small injections of 3′-CMP solution in the same buffer. The enthalpy data were fitted to a single site binding model. ( A ) Binding isotherm for refolded RNase A; ( B ) binding isotherm for Sigma RNase A.

Techniques Used: Binding Assay

Spectroscopic characterization of refolded RNase A. ( A ) Fluorescence emission spectra of RNase A. The samples at a concentration of 100 μg/mL −1 (∼7 μM) were excited at 278 nm and the emission spectra from 290 to 400 nm were recorded, using excitation and emission slit widths of 2 nm and 5 nm, respectively. Fluorescence spectra of unfolded RNase A (3), refolded RNase A (2), Sigma RNase A (1) are shown after correction for buffer contribution. ( B ) The far-UV CD spectra of refolded RNase A (○) and Sigma RNase A (△) for 0.5 mg/mL −1 protein recorded in a 1-mm path length cuvette from 200 to 250 nm. ( C ) The near-UV CD spectra of the two samples recorded for 0.5 mg/mL −1 protein in a 1-cm path length cuvette from 250 to 350 nm. The symbols are the same as in B .
Figure Legend Snippet: Spectroscopic characterization of refolded RNase A. ( A ) Fluorescence emission spectra of RNase A. The samples at a concentration of 100 μg/mL −1 (∼7 μM) were excited at 278 nm and the emission spectra from 290 to 400 nm were recorded, using excitation and emission slit widths of 2 nm and 5 nm, respectively. Fluorescence spectra of unfolded RNase A (3), refolded RNase A (2), Sigma RNase A (1) are shown after correction for buffer contribution. ( B ) The far-UV CD spectra of refolded RNase A (○) and Sigma RNase A (△) for 0.5 mg/mL −1 protein recorded in a 1-mm path length cuvette from 200 to 250 nm. ( C ) The near-UV CD spectra of the two samples recorded for 0.5 mg/mL −1 protein in a 1-cm path length cuvette from 250 to 350 nm. The symbols are the same as in B .

Techniques Used: Fluorescence, Concentration Assay

TPP purification monitored by SDS-PAGE. ( A ) Fifteen percent SDS-PAGE analysis of RNase A inclusion bodies at different steps of TPP; (lane 1 ) unwashed inclusion bodies; (lane 2 ) washed inclusion bodies with 50 mM PBS/pH 7.4/1 mM EDTA containing 2 M urea; (lane 3 ) commercial RNase A (Sigma Chemical Co.); (lane 4 ) refolded RNase A obtained from interfacial precipitate of second TPP; (lane 5 ) interfacial precipitate of first TPP; (lane 6 ) aqueous phase of second TPP. ( B ) Fifteen percent SDS-PAGE analysis of inclusion bodies of CcdB mutants subjected to TPP; (lane 1 ) unwashed inclusion bodies of CcdB-F17P; (lane 2 ) washed inclusion bodies of CcdB-F17P with 50 mM PBS/pH 7.4/0.5% Triton X-100; (lane 3 ) interfacial precipitate of first TPP; (lane 4 ) aqueous phase of second TPP; (lane 5 ) refolded and purified CcdB-F17P by TPP; (lane 6 ) molecular weight marker; (lane 7 ) unwashed inclusion bodies of CcdB-M97K; (lane 8 ) washed inclusion bodies of CcdB-M97K; (lane 9 ) interfacial precipitate of first TPP; (lane 10 ) aqueous phase of second TPP; (lane 11 ) refolded and purified CcdB-M97K by TPP. ( C ) Fifteen percent SDS-PAGE analysis of inclusion bodies of CD4D12 subjected to TPP CD4D12; (lane 1 ) molecular weight marker; (lane 2 ) unwashed inclusion bodies of CD4D12; (lane 3 ) washed inclusion bodies of CD4D12 with 50 mM PBS/pH 7.4/0.5% Triton X-100; (lane 4 ) interfacial precipitate of first TPP; (lane 5 ) aqueous phase of second TPP; (lane 6 ) refolded and purified CD4D12 by TPP; (lane 7 ) refolded and purified CD4D12 after subjected to the 50-kDa polyethersulfone membrane (PALL Lifesciences) once; (lane 8 ) refolded and purified CD4D12 after subjected to the 50-kDa polyethersulfone membrane (PALL Lifesciences) twice; (lane 9 ) refolded and purified CD4D12 in the absence of DTT.
Figure Legend Snippet: TPP purification monitored by SDS-PAGE. ( A ) Fifteen percent SDS-PAGE analysis of RNase A inclusion bodies at different steps of TPP; (lane 1 ) unwashed inclusion bodies; (lane 2 ) washed inclusion bodies with 50 mM PBS/pH 7.4/1 mM EDTA containing 2 M urea; (lane 3 ) commercial RNase A (Sigma Chemical Co.); (lane 4 ) refolded RNase A obtained from interfacial precipitate of second TPP; (lane 5 ) interfacial precipitate of first TPP; (lane 6 ) aqueous phase of second TPP. ( B ) Fifteen percent SDS-PAGE analysis of inclusion bodies of CcdB mutants subjected to TPP; (lane 1 ) unwashed inclusion bodies of CcdB-F17P; (lane 2 ) washed inclusion bodies of CcdB-F17P with 50 mM PBS/pH 7.4/0.5% Triton X-100; (lane 3 ) interfacial precipitate of first TPP; (lane 4 ) aqueous phase of second TPP; (lane 5 ) refolded and purified CcdB-F17P by TPP; (lane 6 ) molecular weight marker; (lane 7 ) unwashed inclusion bodies of CcdB-M97K; (lane 8 ) washed inclusion bodies of CcdB-M97K; (lane 9 ) interfacial precipitate of first TPP; (lane 10 ) aqueous phase of second TPP; (lane 11 ) refolded and purified CcdB-M97K by TPP. ( C ) Fifteen percent SDS-PAGE analysis of inclusion bodies of CD4D12 subjected to TPP CD4D12; (lane 1 ) molecular weight marker; (lane 2 ) unwashed inclusion bodies of CD4D12; (lane 3 ) washed inclusion bodies of CD4D12 with 50 mM PBS/pH 7.4/0.5% Triton X-100; (lane 4 ) interfacial precipitate of first TPP; (lane 5 ) aqueous phase of second TPP; (lane 6 ) refolded and purified CD4D12 by TPP; (lane 7 ) refolded and purified CD4D12 after subjected to the 50-kDa polyethersulfone membrane (PALL Lifesciences) once; (lane 8 ) refolded and purified CD4D12 after subjected to the 50-kDa polyethersulfone membrane (PALL Lifesciences) twice; (lane 9 ) refolded and purified CD4D12 in the absence of DTT.

Techniques Used: Purification, SDS Page, Molecular Weight, Marker

62) Product Images from "Refolding and simultaneous purification by three-phase partitioning of recombinant proteins from inclusion bodies"

Article Title: Refolding and simultaneous purification by three-phase partitioning of recombinant proteins from inclusion bodies

Journal: Protein Science : A Publication of the Protein Society

doi: 10.1110/ps.036939.108

The binding isotherm of RNase A and 3′-CMP. RNase A (55 μM) in 0.2 M sodium acetate buffer, pH 6.0 containing 0.2 M NaCl, was titrated with small injections of 3′-CMP solution in the same buffer. The enthalpy data were fitted to a single site binding model. ( A ) Binding isotherm for refolded RNase A; ( B ) binding isotherm for Sigma RNase A.
Figure Legend Snippet: The binding isotherm of RNase A and 3′-CMP. RNase A (55 μM) in 0.2 M sodium acetate buffer, pH 6.0 containing 0.2 M NaCl, was titrated with small injections of 3′-CMP solution in the same buffer. The enthalpy data were fitted to a single site binding model. ( A ) Binding isotherm for refolded RNase A; ( B ) binding isotherm for Sigma RNase A.

Techniques Used: Binding Assay

Spectroscopic characterization of refolded RNase A. ( A ) Fluorescence emission spectra of RNase A. The samples at a concentration of 100 μg/mL −1 (∼7 μM) were excited at 278 nm and the emission spectra from 290 to 400 nm were recorded, using excitation and emission slit widths of 2 nm and 5 nm, respectively. Fluorescence spectra of unfolded RNase A (3), refolded RNase A (2), Sigma RNase A (1) are shown after correction for buffer contribution. ( B ) The far-UV CD spectra of refolded RNase A (○) and Sigma RNase A (△) for 0.5 mg/mL −1 protein recorded in a 1-mm path length cuvette from 200 to 250 nm. ( C ) The near-UV CD spectra of the two samples recorded for 0.5 mg/mL −1 protein in a 1-cm path length cuvette from 250 to 350 nm. The symbols are the same as in B .
Figure Legend Snippet: Spectroscopic characterization of refolded RNase A. ( A ) Fluorescence emission spectra of RNase A. The samples at a concentration of 100 μg/mL −1 (∼7 μM) were excited at 278 nm and the emission spectra from 290 to 400 nm were recorded, using excitation and emission slit widths of 2 nm and 5 nm, respectively. Fluorescence spectra of unfolded RNase A (3), refolded RNase A (2), Sigma RNase A (1) are shown after correction for buffer contribution. ( B ) The far-UV CD spectra of refolded RNase A (○) and Sigma RNase A (△) for 0.5 mg/mL −1 protein recorded in a 1-mm path length cuvette from 200 to 250 nm. ( C ) The near-UV CD spectra of the two samples recorded for 0.5 mg/mL −1 protein in a 1-cm path length cuvette from 250 to 350 nm. The symbols are the same as in B .

Techniques Used: Fluorescence, Concentration Assay

TPP purification monitored by SDS-PAGE. ( A ) Fifteen percent SDS-PAGE analysis of RNase A inclusion bodies at different steps of TPP; (lane 1 ) unwashed inclusion bodies; (lane 2 ) washed inclusion bodies with 50 mM PBS/pH 7.4/1 mM EDTA containing 2 M urea; (lane 3 ) commercial RNase A (Sigma Chemical Co.); (lane 4 ) refolded RNase A obtained from interfacial precipitate of second TPP; (lane 5 ) interfacial precipitate of first TPP; (lane 6 ) aqueous phase of second TPP. ( B ) Fifteen percent SDS-PAGE analysis of inclusion bodies of CcdB mutants subjected to TPP; (lane 1 ) unwashed inclusion bodies of CcdB-F17P; (lane 2 ) washed inclusion bodies of CcdB-F17P with 50 mM PBS/pH 7.4/0.5% Triton X-100; (lane 3 ) interfacial precipitate of first TPP; (lane 4 ) aqueous phase of second TPP; (lane 5 ) refolded and purified CcdB-F17P by TPP; (lane 6 ) molecular weight marker; (lane 7 ) unwashed inclusion bodies of CcdB-M97K; (lane 8 ) washed inclusion bodies of CcdB-M97K; (lane 9 ) interfacial precipitate of first TPP; (lane 10 ) aqueous phase of second TPP; (lane 11 ) refolded and purified CcdB-M97K by TPP. ( C ) Fifteen percent SDS-PAGE analysis of inclusion bodies of CD4D12 subjected to TPP CD4D12; (lane 1 ) molecular weight marker; (lane 2 ) unwashed inclusion bodies of CD4D12; (lane 3 ) washed inclusion bodies of CD4D12 with 50 mM PBS/pH 7.4/0.5% Triton X-100; (lane 4 ) interfacial precipitate of first TPP; (lane 5 ) aqueous phase of second TPP; (lane 6 ) refolded and purified CD4D12 by TPP; (lane 7 ) refolded and purified CD4D12 after subjected to the 50-kDa polyethersulfone membrane (PALL Lifesciences) once; (lane 8 ) refolded and purified CD4D12 after subjected to the 50-kDa polyethersulfone membrane (PALL Lifesciences) twice; (lane 9 ) refolded and purified CD4D12 in the absence of DTT.
Figure Legend Snippet: TPP purification monitored by SDS-PAGE. ( A ) Fifteen percent SDS-PAGE analysis of RNase A inclusion bodies at different steps of TPP; (lane 1 ) unwashed inclusion bodies; (lane 2 ) washed inclusion bodies with 50 mM PBS/pH 7.4/1 mM EDTA containing 2 M urea; (lane 3 ) commercial RNase A (Sigma Chemical Co.); (lane 4 ) refolded RNase A obtained from interfacial precipitate of second TPP; (lane 5 ) interfacial precipitate of first TPP; (lane 6 ) aqueous phase of second TPP. ( B ) Fifteen percent SDS-PAGE analysis of inclusion bodies of CcdB mutants subjected to TPP; (lane 1 ) unwashed inclusion bodies of CcdB-F17P; (lane 2 ) washed inclusion bodies of CcdB-F17P with 50 mM PBS/pH 7.4/0.5% Triton X-100; (lane 3 ) interfacial precipitate of first TPP; (lane 4 ) aqueous phase of second TPP; (lane 5 ) refolded and purified CcdB-F17P by TPP; (lane 6 ) molecular weight marker; (lane 7 ) unwashed inclusion bodies of CcdB-M97K; (lane 8 ) washed inclusion bodies of CcdB-M97K; (lane 9 ) interfacial precipitate of first TPP; (lane 10 ) aqueous phase of second TPP; (lane 11 ) refolded and purified CcdB-M97K by TPP. ( C ) Fifteen percent SDS-PAGE analysis of inclusion bodies of CD4D12 subjected to TPP CD4D12; (lane 1 ) molecular weight marker; (lane 2 ) unwashed inclusion bodies of CD4D12; (lane 3 ) washed inclusion bodies of CD4D12 with 50 mM PBS/pH 7.4/0.5% Triton X-100; (lane 4 ) interfacial precipitate of first TPP; (lane 5 ) aqueous phase of second TPP; (lane 6 ) refolded and purified CD4D12 by TPP; (lane 7 ) refolded and purified CD4D12 after subjected to the 50-kDa polyethersulfone membrane (PALL Lifesciences) once; (lane 8 ) refolded and purified CD4D12 after subjected to the 50-kDa polyethersulfone membrane (PALL Lifesciences) twice; (lane 9 ) refolded and purified CD4D12 in the absence of DTT.

Techniques Used: Purification, SDS Page, Molecular Weight, Marker

63) Product Images from "Functional topography of nascent RNA in elongation intermediates of RNA polymerase"

Article Title: Functional topography of nascent RNA in elongation intermediates of RNA polymerase

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi:

Analysis of the integrity and catalytic activity of ECs containing RNAs truncated with the RNases T1 and A. The origin of the cleavages introduced in the proximity of the RNA 3′ end. ( A Left ) Lanes 1–6, EC 32 (lane 1) was treated with RNase T1 (lane 2, t, total). To test catalytic activity, the cleaved complex was incubated with UTP (lane 3, t+UTP). To test integrity, the cleaved complex was either washed (lane 4, pw, pellet washed) or divided into pellet and supernatant (lanes 5 and 6, p and s). Phenol was added to all the samples to inactivate the RNases. ( A Center ) Lanes 7–16, EC 32 (lane 7) was treated with two doses of RNase T1. The cleavage was stopped either with phenol (lanes 8 and 10, t) or with 2′-GMP plus phenol (lanes 9 and 11, t+inh). Homogeneous arrested EC 30 was obtained as described in Materials and Methods (lane 12) and treated with two doses of RNase T1. The cleavage was stopped either with phenol (lanes 13 and 15, t) or with 2′-GMP plus phenol (lanes 14 and 16, t+inh). ( A Right ) Lanes 17–23, EC 20 (lane 17) was treated with RNase A and its catalytic activity and integrity were analyzed as described for A Left (lanes 18–22). Lane 23, the cleavage was stopped by Prime RNase Inhibitor plus phenol. The shaded rectangles represent the segments of the RNAs protected by RNAP in the intact ECs. ( B ) Homogeneous arrested EC 30 and EC 32 (lanes 2 and 4) were obtained as described in Materials and Methods ). Lane 1, 20-nt RNA used as a size marker.
Figure Legend Snippet: Analysis of the integrity and catalytic activity of ECs containing RNAs truncated with the RNases T1 and A. The origin of the cleavages introduced in the proximity of the RNA 3′ end. ( A Left ) Lanes 1–6, EC 32 (lane 1) was treated with RNase T1 (lane 2, t, total). To test catalytic activity, the cleaved complex was incubated with UTP (lane 3, t+UTP). To test integrity, the cleaved complex was either washed (lane 4, pw, pellet washed) or divided into pellet and supernatant (lanes 5 and 6, p and s). Phenol was added to all the samples to inactivate the RNases. ( A Center ) Lanes 7–16, EC 32 (lane 7) was treated with two doses of RNase T1. The cleavage was stopped either with phenol (lanes 8 and 10, t) or with 2′-GMP plus phenol (lanes 9 and 11, t+inh). Homogeneous arrested EC 30 was obtained as described in Materials and Methods (lane 12) and treated with two doses of RNase T1. The cleavage was stopped either with phenol (lanes 13 and 15, t) or with 2′-GMP plus phenol (lanes 14 and 16, t+inh). ( A Right ) Lanes 17–23, EC 20 (lane 17) was treated with RNase A and its catalytic activity and integrity were analyzed as described for A Left (lanes 18–22). Lane 23, the cleavage was stopped by Prime RNase Inhibitor plus phenol. The shaded rectangles represent the segments of the RNAs protected by RNAP in the intact ECs. ( B ) Homogeneous arrested EC 30 and EC 32 (lanes 2 and 4) were obtained as described in Materials and Methods ). Lane 1, 20-nt RNA used as a size marker.

Techniques Used: Activity Assay, Incubation, Marker

Cleavage of the RNAs with RNases T1 and A in a number of consecutive ECs and their effect on EC stability. ( A ) The indicated ECs were digested with 5,000 units/ml of RNase T1 ( Left ) or with 5 μg/ml RNase A ( Right ), washed, and then combined with phenol. ( B ) Summary of RNase footprinting data obtained for the T7A1 transcription unit. Arrows show the cleavage sites introduced in the RNAs at high doses of RNases T1 and A, when the cleavage was stopped either by adding nondenaturing inhibitors or by washing off the RNases. The information shown is based on 3–5 separate experiments performed with RNAs labeled at various positions. The shaded rectangles represent the minimal 14-nt segment of the RNAs protected by RNAP in the intact ECs. ( C ) The indicated ECs, either intact or treated with RNase T1 and washed as described in A , were incubated in TB containing 300 mM KCl before separating the samples into supernatant and pellet (s and p).
Figure Legend Snippet: Cleavage of the RNAs with RNases T1 and A in a number of consecutive ECs and their effect on EC stability. ( A ) The indicated ECs were digested with 5,000 units/ml of RNase T1 ( Left ) or with 5 μg/ml RNase A ( Right ), washed, and then combined with phenol. ( B ) Summary of RNase footprinting data obtained for the T7A1 transcription unit. Arrows show the cleavage sites introduced in the RNAs at high doses of RNases T1 and A, when the cleavage was stopped either by adding nondenaturing inhibitors or by washing off the RNases. The information shown is based on 3–5 separate experiments performed with RNAs labeled at various positions. The shaded rectangles represent the minimal 14-nt segment of the RNAs protected by RNAP in the intact ECs. ( C ) The indicated ECs, either intact or treated with RNase T1 and washed as described in A , were incubated in TB containing 300 mM KCl before separating the samples into supernatant and pellet (s and p).

Techniques Used: Footprinting, Labeling, Incubation

Analysis of the binding of RNAP to RNA upstream from the protected zone. The cleavage of EC 30 with 5 μg/ml of RNase A was stopped with phenol in the presence of Prime RNase Inhibitor. Products of the cleavage (t) were either fractionated into supernatant and pellet (s and p) or washed with TB (p w ) as described in Materials and Methods .
Figure Legend Snippet: Analysis of the binding of RNAP to RNA upstream from the protected zone. The cleavage of EC 30 with 5 μg/ml of RNase A was stopped with phenol in the presence of Prime RNase Inhibitor. Products of the cleavage (t) were either fractionated into supernatant and pellet (s and p) or washed with TB (p w ) as described in Materials and Methods .

Techniques Used: Binding Assay

64) Product Images from "Generation of a Novel Nucleic Acid-Based Reporter System To Detect Phenotypic Susceptibility to Antibiotics in Mycobacterium tuberculosis"

Article Title: Generation of a Novel Nucleic Acid-Based Reporter System To Detect Phenotypic Susceptibility to Antibiotics in Mycobacterium tuberculosis

Journal: mBio

doi: 10.1128/mBio.00312-11

SML RNA present in phSP6-ProPol stocks. A crude phSP6-ProPol preparation (10 8 PFU) was either left untreated or treated with MRI to a final concentration of 1 U/μl. Either 5 ng (++) or 1 ng (+) RNase A was then added, and the mixture was incubated for 30 min at 37°C. After purification of RNA and digestion with DNase I, reverse transcription was carried out using the DnSt primer. After reverse transcription but prior to PCR, cDNA from each sample was diluted 1:10 to estimate a 10-fold signal reduction. The primers DnSt and UpSt were then used to amplify a 150-bp section of SML cDNA using PCR. Products were separated on a 2% agarose gel and visualized by ethidium bromide staining. The locations of DNA size markers are indicated.
Figure Legend Snippet: SML RNA present in phSP6-ProPol stocks. A crude phSP6-ProPol preparation (10 8 PFU) was either left untreated or treated with MRI to a final concentration of 1 U/μl. Either 5 ng (++) or 1 ng (+) RNase A was then added, and the mixture was incubated for 30 min at 37°C. After purification of RNA and digestion with DNase I, reverse transcription was carried out using the DnSt primer. After reverse transcription but prior to PCR, cDNA from each sample was diluted 1:10 to estimate a 10-fold signal reduction. The primers DnSt and UpSt were then used to amplify a 150-bp section of SML cDNA using PCR. Products were separated on a 2% agarose gel and visualized by ethidium bromide staining. The locations of DNA size markers are indicated.

Techniques Used: Magnetic Resonance Imaging, Concentration Assay, Incubation, Purification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining

SML detection occurs at 4 h postinfection (p.i.). H37Rv was infected with phSP6-ProPol. RNase A was added to phSP6-ProPol prior to initiation of infection, and MRI was added at 0.5 h p.i. At 0.5, 3, and 4 h p.i., RNA was purified, digested with DNase I, and amplified using RT-PCR. Products were then separated on a 2% agarose gel and visualized by ethidium bromide staining. The locations of DNA size markers are indicated.
Figure Legend Snippet: SML detection occurs at 4 h postinfection (p.i.). H37Rv was infected with phSP6-ProPol. RNase A was added to phSP6-ProPol prior to initiation of infection, and MRI was added at 0.5 h p.i. At 0.5, 3, and 4 h p.i., RNA was purified, digested with DNase I, and amplified using RT-PCR. Products were then separated on a 2% agarose gel and visualized by ethidium bromide staining. The locations of DNA size markers are indicated.

Techniques Used: Infection, Magnetic Resonance Imaging, Purification, Amplification, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining

65) Product Images from "Replacing a single atom accelerates the folding of a protein and increases its thermostability"

Article Title: Replacing a single atom accelerates the folding of a protein and increases its thermostability

Journal: Organic & biomolecular chemistry

doi: 10.1039/c6ob00980h

Kinetics of the unfolding and folding of wild-type RNase A and its Pro114→flp variant. (A) Arrhenius plot of unfolding as determined by limited proteolysis with thermolysin. (B) Arrhenius plot of folding as determined by the gain of enzymatic activity.
Figure Legend Snippet: Kinetics of the unfolding and folding of wild-type RNase A and its Pro114→flp variant. (A) Arrhenius plot of unfolding as determined by limited proteolysis with thermolysin. (B) Arrhenius plot of folding as determined by the gain of enzymatic activity.

Techniques Used: Variant Assay, Activity Assay

Circular dichroism spectra of wild-type RNase A and its Pro114→flp variant. (A) Far-UV region. (B) Near-UV region.
Figure Legend Snippet: Circular dichroism spectra of wild-type RNase A and its Pro114→flp variant. (A) Far-UV region. (B) Near-UV region.

Techniques Used: Variant Assay

66) Product Images from "Functional and structural analyses of N-acylsulfonamide-linked dinucleoside inhibitors of RNase A"

Article Title: Functional and structural analyses of N-acylsulfonamide-linked dinucleoside inhibitors of RNase A

Journal: The Febs Journal

doi: 10.1111/j.1742-4658.2010.07976.x

(A, B) Schematic and stereo representation of hydrogen bonds in the RNase A complex with N -acylsulfonamide 7 and N -acylsulfonamide 6 , respectively. N -Acylsulfonamide 7 and N -acylsulfonamide 6 , gold; active site residues, pea-green; RNase A, gray. Hydrogen bonds are represented as dashed lines, and water molecules are in cyan. (C, D) Stereo pictures of 2 F o − F c contoured at 1.0 σ for N -acylsulfonamide 7 and N -acylsulfonamide 6 , respectively.
Figure Legend Snippet: (A, B) Schematic and stereo representation of hydrogen bonds in the RNase A complex with N -acylsulfonamide 7 and N -acylsulfonamide 6 , respectively. N -Acylsulfonamide 7 and N -acylsulfonamide 6 , gold; active site residues, pea-green; RNase A, gray. Hydrogen bonds are represented as dashed lines, and water molecules are in cyan. (C, D) Stereo pictures of 2 F o − F c contoured at 1.0 σ for N -acylsulfonamide 7 and N -acylsulfonamide 6 , respectively.

Techniques Used:

Isotherms for the binding of  N -acylsulfonamide-linked dinucleosides to RNase A. Data were fitted to   Eqn (1) . (A)  N -acylsulfonamide  7 ,  K i  = (3.7 ± 0.1) × 10 −4 m . (B)  N -acylsulfonamide  6 ,  K i  = (4.6 ± 0.3) × 10 −4 m .
Figure Legend Snippet: Isotherms for the binding of N -acylsulfonamide-linked dinucleosides to RNase A. Data were fitted to Eqn (1) . (A) N -acylsulfonamide 7 , K i = (3.7 ± 0.1) × 10 −4 m . (B) N -acylsulfonamide 6 , K i = (4.6 ± 0.3) × 10 −4 m .

Techniques Used: Binding Assay

67) Product Images from "RNA-dependent cytoplasmic anchoring of a transcription factor subunit during Xenopus development"

Article Title: RNA-dependent cytoplasmic anchoring of a transcription factor subunit during Xenopus development

Journal: The EMBO Journal

doi: 10.1093/emboj/19.14.3683

Fig. 7. RNA acts as a cytoplasmic anchor for CBTF 122 . ( A ) An additional NLS does not make CBTF 122 nuclear during early development. Two-cell embryos from a single female Xenopus were injected with 200 pg of synthetic RNA encoding the proteins shown into the animal pole of each cell and allowed to develop. At stage 8, embryos injected with RNA encoding the full-length myc-tagged proteins were fixed and exogenous protein visualized by immunohistochemistry of the myc tag (top panels). For embryos expressing the GFP constructs, animal pole explants were removed and squashed gently under a coverslip in the presence of DAPI. This released nuclei from the surrounding, intrinsically fluorescent yolk granules. GFP and DAPI staining were then visualized by fluorescence microscopy. ( B ) Degradation of endogenous RNA leads to nuclear translocation of CBTF 122 before the MBT. Embryos from a single female Xenopus were taken at stage 6 and a single blastomere injected with 1 nl of either buffer (control) or buffer containing 50 ng of RNase A (+RNase). After incubation for a further 60 min, cell division was arrested by incubation in cycloheximide. Embryos were fixed, endogenous CBTF 122/98 was visualized by immunohistochemistry and the embryos cleared. The cell receiving the RNase injection is markedly larger than those around it and shows nuclear staining (black arrow), as do some surrounding cells that also received RNase. The white arrow shows perinuclear staining, which has been observed previously at these early stages. Only limited perinuclear staining is observed in controls.
Figure Legend Snippet: Fig. 7. RNA acts as a cytoplasmic anchor for CBTF 122 . ( A ) An additional NLS does not make CBTF 122 nuclear during early development. Two-cell embryos from a single female Xenopus were injected with 200 pg of synthetic RNA encoding the proteins shown into the animal pole of each cell and allowed to develop. At stage 8, embryos injected with RNA encoding the full-length myc-tagged proteins were fixed and exogenous protein visualized by immunohistochemistry of the myc tag (top panels). For embryos expressing the GFP constructs, animal pole explants were removed and squashed gently under a coverslip in the presence of DAPI. This released nuclei from the surrounding, intrinsically fluorescent yolk granules. GFP and DAPI staining were then visualized by fluorescence microscopy. ( B ) Degradation of endogenous RNA leads to nuclear translocation of CBTF 122 before the MBT. Embryos from a single female Xenopus were taken at stage 6 and a single blastomere injected with 1 nl of either buffer (control) or buffer containing 50 ng of RNase A (+RNase). After incubation for a further 60 min, cell division was arrested by incubation in cycloheximide. Embryos were fixed, endogenous CBTF 122/98 was visualized by immunohistochemistry and the embryos cleared. The cell receiving the RNase injection is markedly larger than those around it and shows nuclear staining (black arrow), as do some surrounding cells that also received RNase. The white arrow shows perinuclear staining, which has been observed previously at these early stages. Only limited perinuclear staining is observed in controls.

Techniques Used: Injection, Immunohistochemistry, Expressing, Construct, Staining, Fluorescence, Microscopy, Translocation Assay, Incubation

Fig. 3. CBTF 122 and CBTF 98 bind RNA in vitro and are associated with non-translating mRNAs in cleavage stage embryos. ( A ) Northwestern blot analysis of total soluble proteins from two stage VI oocytes (VI) or two embryos at the stages shown. Proteins were separated by SDS–PAGE and transferred to nitrocellulose. The blot was probed first with 32 P-labelled L1 5′-UTR (northwestern) and subsequently probed with affinity-purified anti-CBTF 122/98 antibody (western). ( B ) Cytoplasmic proteins from cleavage-stage embryos were incubated in the absence or presence of RNase A and T1 as indicated and then sedimented through a 20–60% nycodenz gradient. Protein precipitated from the indicated fractions was analysed by western blotting using affinity-purified anti-CBTF 122/98 antibody or anti-mRNP3+4 antibody. ( C ) Fractions 12 and 13 of the (–)RNase gradient were mixed and aliquots were incubated in the absence or presence of RNase A and T1. The RNase-treated or untreated aliquots were incubated with anti-mRNP3+4 antiserum (α-mRNP3+4) and protein A beads as shown. Western blots of the supernatant (S) and pellet (P) fractions were probed with affinity-purified anti-p122/p98 (upper panel) or anti-mRNP3+4 (lower panel) antibodies.
Figure Legend Snippet: Fig. 3. CBTF 122 and CBTF 98 bind RNA in vitro and are associated with non-translating mRNAs in cleavage stage embryos. ( A ) Northwestern blot analysis of total soluble proteins from two stage VI oocytes (VI) or two embryos at the stages shown. Proteins were separated by SDS–PAGE and transferred to nitrocellulose. The blot was probed first with 32 P-labelled L1 5′-UTR (northwestern) and subsequently probed with affinity-purified anti-CBTF 122/98 antibody (western). ( B ) Cytoplasmic proteins from cleavage-stage embryos were incubated in the absence or presence of RNase A and T1 as indicated and then sedimented through a 20–60% nycodenz gradient. Protein precipitated from the indicated fractions was analysed by western blotting using affinity-purified anti-CBTF 122/98 antibody or anti-mRNP3+4 antibody. ( C ) Fractions 12 and 13 of the (–)RNase gradient were mixed and aliquots were incubated in the absence or presence of RNase A and T1. The RNase-treated or untreated aliquots were incubated with anti-mRNP3+4 antiserum (α-mRNP3+4) and protein A beads as shown. Western blots of the supernatant (S) and pellet (P) fractions were probed with affinity-purified anti-p122/p98 (upper panel) or anti-mRNP3+4 (lower panel) antibodies.

Techniques Used: In Vitro, SDS Page, Affinity Purification, Western Blot, Incubation

68) Product Images from "m6A RNA methylation regulates the UV-induced DNA damage response"

Article Title: m6A RNA methylation regulates the UV-induced DNA damage response

Journal: Nature

doi: 10.1038/nature21671

6 A-modified RNA accumulates at damage sites in response to UV irradiation a , U2OS cells were subjected to the indicated doses of UVC irradiation, incubated at 37°C for 2 min, and costained for m 6 A and γH2A.X. Relative m 6 A intensity is indicated on the right. b , U2OS cells were subjected to 50 J UVC irradiation through a micropore filter, incubated at 37°C for 2 or 5 min, and costained for m 6 A and DNA (DAPI). c , A375 (melanoma) or HeLa cells were subjected or not (0′) to 25 J UVC irradiation, incubated at 37°C for the indicated times, and then costained for m 6 A and γH2A.X. d, U2OS cells were subjected or not (0′) to 20 Gray of γ-irradiation, incubated at 37°C for 4 min, then costained for m 6 A and γH2A.X. e , U2OS cells were either irradiated with 25 J UVC or treated with DMSO, mitomycin C, hydroxyurea, or arabinoside-C, then costained for m 6 A and γH2A.X. c–e , relative m 6 A intensity is indicated on the right. f , Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) cells were subjected to 25 J UVC irradiation (top panel) or microirradiation by UVA laser (bottom panel), incubated at 37°C for 2 min, and stained for m 6 A. Top and bottom panels show m 6 A signal in representative S/G2/M or G1 phase cells, respectively. Arrows in middle panels denote representative G1-phase cells negative for m 6 A signal. The percentage of G1 (red) or S/G2/M (green) cells positive for m 6 A signal is indicated on the right. g , U2OS cells were microirradiated by UVA laser, permeabilized and treated with or without RNase A, and costained for m 6 A and γH2A.X (left panel). The percentage of γH2A.X-positive cells displaying colocalizing m 6 A signal is indicated. Nucleic acids (DNA and RNA) from cells treated with or without RNase A were isolated and analyzed on an agarose gel (right panel). Ladder (1kb DNA ladder). h , Poly(A)+ RNA was extracted from the samples in (a), and subjected to dot-blot analysis with an antibody recognizing m 6 A. Methylene blue staining was used as a loading control.
Figure Legend Snippet: 6 A-modified RNA accumulates at damage sites in response to UV irradiation a , U2OS cells were subjected to the indicated doses of UVC irradiation, incubated at 37°C for 2 min, and costained for m 6 A and γH2A.X. Relative m 6 A intensity is indicated on the right. b , U2OS cells were subjected to 50 J UVC irradiation through a micropore filter, incubated at 37°C for 2 or 5 min, and costained for m 6 A and DNA (DAPI). c , A375 (melanoma) or HeLa cells were subjected or not (0′) to 25 J UVC irradiation, incubated at 37°C for the indicated times, and then costained for m 6 A and γH2A.X. d, U2OS cells were subjected or not (0′) to 20 Gray of γ-irradiation, incubated at 37°C for 4 min, then costained for m 6 A and γH2A.X. e , U2OS cells were either irradiated with 25 J UVC or treated with DMSO, mitomycin C, hydroxyurea, or arabinoside-C, then costained for m 6 A and γH2A.X. c–e , relative m 6 A intensity is indicated on the right. f , Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) cells were subjected to 25 J UVC irradiation (top panel) or microirradiation by UVA laser (bottom panel), incubated at 37°C for 2 min, and stained for m 6 A. Top and bottom panels show m 6 A signal in representative S/G2/M or G1 phase cells, respectively. Arrows in middle panels denote representative G1-phase cells negative for m 6 A signal. The percentage of G1 (red) or S/G2/M (green) cells positive for m 6 A signal is indicated on the right. g , U2OS cells were microirradiated by UVA laser, permeabilized and treated with or without RNase A, and costained for m 6 A and γH2A.X (left panel). The percentage of γH2A.X-positive cells displaying colocalizing m 6 A signal is indicated. Nucleic acids (DNA and RNA) from cells treated with or without RNase A were isolated and analyzed on an agarose gel (right panel). Ladder (1kb DNA ladder). h , Poly(A)+ RNA was extracted from the samples in (a), and subjected to dot-blot analysis with an antibody recognizing m 6 A. Methylene blue staining was used as a loading control.

Techniques Used: Modification, Irradiation, Incubation, Staining, Isolation, Agarose Gel Electrophoresis, Dot Blot

69) Product Images from "Phosphorothioate Antisense Oligonucleotides Induce the Formation of Nuclear Bodies"

Article Title: Phosphorothioate Antisense Oligonucleotides Induce the Formation of Nuclear Bodies

Journal: Molecular Biology of the Cell

doi:

Comparative measurements of cell-based PS-ONs by indirect enzyme immunoassay corroborated the binding of a major amount of the oligonucleotides to the nuclear matrix. (A) HeLa cells grown in 96-well plates were lipofected with 0.25 μM 11068-F, fixed directly (fixed) or fractionated consecutively with CSK buffer (CSK), extraction buffer (high salt), and DNase (DNase). There was little loss during fractionation. (B) Experiment in which the DNase step had been substituted with buffer alone (buffer) or RNase A (RNase). RNase treatment resulted in a strong displacement of PS-ONs from the nuclear remnants. The values represent the means ± SD of six wells of a representative experiment corrected for the means obtained for cells that had not been lipofected with oligonucleotide. They were normalized against the mean obtained for directly fixed cells (A) or buffer-treated cells (B), which was set at 100%. Similar results were obtained with a different PS-ON.
Figure Legend Snippet: Comparative measurements of cell-based PS-ONs by indirect enzyme immunoassay corroborated the binding of a major amount of the oligonucleotides to the nuclear matrix. (A) HeLa cells grown in 96-well plates were lipofected with 0.25 μM 11068-F, fixed directly (fixed) or fractionated consecutively with CSK buffer (CSK), extraction buffer (high salt), and DNase (DNase). There was little loss during fractionation. (B) Experiment in which the DNase step had been substituted with buffer alone (buffer) or RNase A (RNase). RNase treatment resulted in a strong displacement of PS-ONs from the nuclear remnants. The values represent the means ± SD of six wells of a representative experiment corrected for the means obtained for cells that had not been lipofected with oligonucleotide. They were normalized against the mean obtained for directly fixed cells (A) or buffer-treated cells (B), which was set at 100%. Similar results were obtained with a different PS-ON.

Techniques Used: Enzyme-linked Immunosorbent Assay, Binding Assay, Fractionation

Nuclear fractionation suggested that the majority of intranuclear PS-ONs resided at the nuclear matrix as the nuclear binding was resistant to detergent, salt, and DNase but not to RNase treatment. HeLa cells lipofected with 0.25 μM PS-ON 11068-F were either directly fixed (A) or underwent consecutive treatments with CSK buffer containing 0.5% Triton X-100 (B), extraction buffer containing high salt concentration (C and D) and DNase (E and F) before they were fixed. DAPI staining verified the digestion of the DNA (D vs. F). Cells were also treated with buffer alone (G) or RNase A (H) instead of DNase. Epifluorescence images of the oligonucleotide signals (A–C, E, G, and H) and DNA signals (D and F) were taken from samples of the same lipofection and were acquired and processed in the same manner for panels A–C and E; panels D and F; panels G and H. Bar, 5 μm.
Figure Legend Snippet: Nuclear fractionation suggested that the majority of intranuclear PS-ONs resided at the nuclear matrix as the nuclear binding was resistant to detergent, salt, and DNase but not to RNase treatment. HeLa cells lipofected with 0.25 μM PS-ON 11068-F were either directly fixed (A) or underwent consecutive treatments with CSK buffer containing 0.5% Triton X-100 (B), extraction buffer containing high salt concentration (C and D) and DNase (E and F) before they were fixed. DAPI staining verified the digestion of the DNA (D vs. F). Cells were also treated with buffer alone (G) or RNase A (H) instead of DNase. Epifluorescence images of the oligonucleotide signals (A–C, E, G, and H) and DNA signals (D and F) were taken from samples of the same lipofection and were acquired and processed in the same manner for panels A–C and E; panels D and F; panels G and H. Bar, 5 μm.

Techniques Used: Fractionation, Binding Assay, Concentration Assay, Staining

70) Product Images from "DBIRD integrates alternative mRNA splicing with RNA polymerase II transcript elongation"

Article Title: DBIRD integrates alternative mRNA splicing with RNA polymerase II transcript elongation

Journal: Nature

doi: 10.1038/nature10925

Purification of nascent nuclear mRNP particles ( a ) Western of cytoplasmn (C), nucleoplasmn (N), and chromatin (Ch), with α-tubulin, lamin B2, and histone H3 as controls for different fractions. ( b ) Fractionation as in (a), but RNAse A added to the nuclear lysis buffer where indicated. ( c ) Purification procedure outline. ( d ) Equal amount of the M2 chromatography eluates from control (Mock) and A1-Flag separated by 4-12% SDS-PAGE, stained with Sypro ruby. Arrow indicates A1-Flag, and asterisks mark RNAse inhibitor proteins. Some identified proteins indicated on the right.
Figure Legend Snippet: Purification of nascent nuclear mRNP particles ( a ) Western of cytoplasmn (C), nucleoplasmn (N), and chromatin (Ch), with α-tubulin, lamin B2, and histone H3 as controls for different fractions. ( b ) Fractionation as in (a), but RNAse A added to the nuclear lysis buffer where indicated. ( c ) Purification procedure outline. ( d ) Equal amount of the M2 chromatography eluates from control (Mock) and A1-Flag separated by 4-12% SDS-PAGE, stained with Sypro ruby. Arrow indicates A1-Flag, and asterisks mark RNAse inhibitor proteins. Some identified proteins indicated on the right.

Techniques Used: Purification, Western Blot, Fractionation, Lysis, Chromatography, SDS Page, Staining

71) Product Images from "Resveratrol directly targets DDX5 resulting in suppression of the mTORC1 pathway in prostate cancer"

Article Title: Resveratrol directly targets DDX5 resulting in suppression of the mTORC1 pathway in prostate cancer

Journal: Cell Death & Disease

doi: 10.1038/cddis.2016.114

Identification of resveratrol-binding proteins. ( a ) The scheme for fixation of resveratrol onto magnetic FG beads with epoxy linkers. ( b ) Purified recombinant PDE4A (2 μ g) was incubated with empty (−) or resveratrol-immobilized (+) beads for 4 h, and bound PDE4A was detected by western blotting. The input lane corresponds to recombinant PDE4A (250 ng). ( c ) Resveratrol-binding proteins were purified from human prostate cancer PC-3 cell extracts, silver-stained, and identified by matrix assisted laser desorption/ionization time-of-flight mass spectrometric analysis. The input lane represents 1% of the PC-3 cell extracts used for the binding assay. ( d ) In the competitive assay, PC-3 cell extracts were preincubated with the indicated doses of resveratrol for 1 h and incubated with the beads for 15 min. Bound DDX5 was detected by western blotting. The input lane represents 5% of the PC-3 cell extracts used for the binding assay. ( e ) Purified recombinant DDX5 (1 μ g) with or without RNase A was incubated with the beads, and bound DDX5 was detected by western blotting. The input lane corresponds to recombinant DDX5 protein (150 ng)
Figure Legend Snippet: Identification of resveratrol-binding proteins. ( a ) The scheme for fixation of resveratrol onto magnetic FG beads with epoxy linkers. ( b ) Purified recombinant PDE4A (2 μ g) was incubated with empty (−) or resveratrol-immobilized (+) beads for 4 h, and bound PDE4A was detected by western blotting. The input lane corresponds to recombinant PDE4A (250 ng). ( c ) Resveratrol-binding proteins were purified from human prostate cancer PC-3 cell extracts, silver-stained, and identified by matrix assisted laser desorption/ionization time-of-flight mass spectrometric analysis. The input lane represents 1% of the PC-3 cell extracts used for the binding assay. ( d ) In the competitive assay, PC-3 cell extracts were preincubated with the indicated doses of resveratrol for 1 h and incubated with the beads for 15 min. Bound DDX5 was detected by western blotting. The input lane represents 5% of the PC-3 cell extracts used for the binding assay. ( e ) Purified recombinant DDX5 (1 μ g) with or without RNase A was incubated with the beads, and bound DDX5 was detected by western blotting. The input lane corresponds to recombinant DDX5 protein (150 ng)

Techniques Used: Binding Assay, Purification, Recombinant, Incubation, Western Blot, Staining

Related Articles

Clone Assay:

Article Title: Determinants of R-loop formation at convergent bidirectionally transcribed trinucleotide repeats
Article Snippet: Plasmids were subsequently treated with RNase A and T1 (Sigma), phenol extracted and purified twice by cesium chloride/ethidium bromide centrifugation and stored in TE (10 mM Tris, 1 mM EDTA pH 7.6) at −20°C. .. Plasmids containing (CGG) · (CCG) and (CAG) · (CTG) repeats were derived from FRAXA, SCA1 and DM1 patients, respectively, and were cloned into pPCRscript-AMP, pBluescript KS(+) or pGEM3Zf(+) ( ).

Centrifugation:

Article Title: Analysis of the oligomeric states of nucleophosmin using size exclusion chromatography
Article Snippet: The suspension was sonicated using a Handy Sonic (TOMY SEICO, Tokyo, Japan) at a power control setting of 5 for 20 sec. After centrifugation at 20,000 × g for 5 min at 4 °C, the supernatant was used as the ‘Triton X-100 insoluble fraction’. .. For RNase A treatment, cell lysates were treated with 40 µg/ml RNase A (Sigma-Aldrich) for 15 min at 37 °C.

Article Title: Determinants of R-loop formation at convergent bidirectionally transcribed trinucleotide repeats
Article Snippet: .. Plasmids were subsequently treated with RNase A and T1 (Sigma), phenol extracted and purified twice by cesium chloride/ethidium bromide centrifugation and stored in TE (10 mM Tris, 1 mM EDTA pH 7.6) at −20°C. .. Plasmids containing (CGG) · (CCG) and (CAG) · (CTG) repeats were derived from FRAXA, SCA1 and DM1 patients, respectively, and were cloned into pPCRscript-AMP, pBluescript KS(+) or pGEM3Zf(+) ( ).

Filtration:

Article Title: A Hinge Region Cis-proline in Ribonuclease A Acts as a Conformational Gatekeeper for C-terminal Domain Swapping
Article Snippet: .. The column was calibrated using a gel filtration molecular weight standard from Biorad and purified RNase A from Sigma. ..

Construct:

Article Title: Conserved amino acid networks modulate discrete functional properties in an enzyme superfamily
Article Snippet: .. DNA constructs, expression and purification DNA sequences of RNase A and human RNase 3 (Eosinophil Cationic Protein) were Escherichia coli codon-optimized, and subcloned into Nde I/Hind III-digested expression vector pET22b(+) (EMD Biosciences, San Diego, CA, USA). .. DNA sequences of human RNase 2 (Eosinophil Derived-Neurotoxin), RNase 4 and RNase 5 (Angiogenin) were acquired from UniProt, Escherichia coli codon-optimized, and subcloned into Nde I/Hind III-digested expression vector pJexpress411/414 (DNA2.0, Menlo Park, CA, USA).

Adsorption:

Article Title: Adsorption-Induced Changes in Ribonuclease A Structure and Enzymatic Activity on Solid Surfaces
Article Snippet: .. II.b Protein Adsorption and Equilibration The adsorption of RNAse A (Sigma R6513) on the material surfaces was carried out using previously described methods (see S.1.a in the ). .. Briefly, 10 mM potassium phosphate buffer solution (PPB; pH 7.4) was prepared by mixing appropriate amounts of 1 M monobasic potassium phosphate (Sigma, P8708) or 1 M dibasic potassium phosphate (Sigma, P8508), following which the buffer concentration was verified by titrating against 0.065 M potassium hydrogen phthalate.

Article Title: Adsorption-Induced Changes in Ribonuclease A Structure and Enzymatic Activity on Solid Surfaces
Article Snippet: .. Briefly, ribonucleic acid, which is the substrate for RNase A, was prepared in PPB to a final concentration of 20 mg/mL (Baker’s yeast, Sigma R6750) and exposed to RNase A both in solution and following RNase A adsorption. .. An initial calibration plot for solution-state enzymatic activity was obtained for a working range of 0.1–30 μg of RNAse A (based on the equivalently adsorbed amount of protein on different surfaces) by monitoring the absorbance at 300 nm (ΔA 300 ) at pH 7.4.

SDS-Gel:

Article Title: Nickel affects xylem Sap RNase a and converts RNase A to a urease
Article Snippet: Purified protein was denatured in SDS-gel sample buffer and electrophoresed on a SDS-10 to 20% polyacrylamide gradient gel. .. The nuclease activity of RNase A from bovine pancreas was verified by suspending RNA (ribonucleic acid from baker’s yeast, Saccharomyces cereviae; Sigma, St. Louis, Mo, USA) in Buffer E [25 mM 2-morpholineethanesulfonic acid (Mes), pH 6.2, 2.5 mM MnSO4 , and 2.5 mM DTT) [ ].

Nuclear Magnetic Resonance:

Article Title: Conserved amino acid networks modulate discrete functional properties in an enzyme superfamily
Article Snippet: DNA constructs, expression and purification DNA sequences of RNase A and human RNase 3 (Eosinophil Cationic Protein) were Escherichia coli codon-optimized, and subcloned into Nde I/Hind III-digested expression vector pET22b(+) (EMD Biosciences, San Diego, CA, USA). .. For NMR experiments, 15 N-labeled samples were prepared by growing E. coli BL21(DE3) in M9 minimal medium supplemented with non-essential amino acids (Life Technologies, Burlington, ON, Canada), metals, glucose, and 15 N-labeled ammonium acetate (Sigma-Aldrich, Oakville, ON, Canada).

Activity Assay:

Article Title: Nickel affects xylem Sap RNase a and converts RNase A to a urease
Article Snippet: .. The nuclease activity of RNase A from bovine pancreas was verified by suspending RNA (ribonucleic acid from baker’s yeast, Saccharomyces cereviae; Sigma, St. Louis, Mo, USA) in Buffer E [25 mM 2-morpholineethanesulfonic acid (Mes), pH 6.2, 2.5 mM MnSO4 , and 2.5 mM DTT) [ ]. .. RNase A (10 μL) was then added to the RNA suspension and the reaction mixture (2 mg/ml) incubated at 25°C for 1 h and then centrifuged (5,500g at 4°C).

Article Title: Adsorption-Induced Changes in Ribonuclease A Structure and Enzymatic Activity on Solid Surfaces
Article Snippet: Paragraph title: Characterization of Enzymatic Activity ... Briefly, ribonucleic acid, which is the substrate for RNase A, was prepared in PPB to a final concentration of 20 mg/mL (Baker’s yeast, Sigma R6750) and exposed to RNase A both in solution and following RNase A adsorption.

Expressing:

Article Title: Conserved amino acid networks modulate discrete functional properties in an enzyme superfamily
Article Snippet: .. DNA constructs, expression and purification DNA sequences of RNase A and human RNase 3 (Eosinophil Cationic Protein) were Escherichia coli codon-optimized, and subcloned into Nde I/Hind III-digested expression vector pET22b(+) (EMD Biosciences, San Diego, CA, USA). .. DNA sequences of human RNase 2 (Eosinophil Derived-Neurotoxin), RNase 4 and RNase 5 (Angiogenin) were acquired from UniProt, Escherichia coli codon-optimized, and subcloned into Nde I/Hind III-digested expression vector pJexpress411/414 (DNA2.0, Menlo Park, CA, USA).

Article Title: Analysis of the oligomeric states of nucleophosmin using size exclusion chromatography
Article Snippet: Preparation of cell lysat To investigate the epitopes of NPM antibody, 293T cells exogenously expressing NPM were lysed with SDS-sample buffer by heating 100 °C for 3 min. For fractionation of the HeLa and 293T cells, cells were lysed with PBS containing 1% Triton X-100. .. For RNase A treatment, cell lysates were treated with 40 µg/ml RNase A (Sigma-Aldrich) for 15 min at 37 °C.

Transplantation Assay:

Article Title: MicroRNA Drop in the Bloodstream and MicroRNA Boost in the Tumour Caused by Treatment with Ribonuclease A Leads to an Attenuation of Tumour Malignancy
Article Snippet: .. On day 4 after tumour transplantation, mice were treated with saline buffer (n = 30) or RNase A (Sigma, USA) at a dose of 0.7 µg/kg (n = 20). ..

Western Blot:

Article Title: Electrolytic Reduction: Modification of Proteins Occurring in Isoelectric Focusing Electrophoresis and in Electrolytic Reactions in the Presence of High Salts
Article Snippet: .. Acrylamido buffers, 0.2 M stock solution (Fluka); GelBond PAG film (BioWhittaker); ribonuclease A, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), bovine insulin, and ovalbumin (Sigma); phenol red (Merck); low-ranged molecular weight markers (Calbiochem); EZ-Link Pentylamine-Biotin, EZ-Link biotin-LC-hydrazide, GelCode blue dye solution, and horseradish peroxidase-conjugated streptavidin (Thermo Scientific); OxyBlot Protein Oxidation Detection Kit (Chemicon); Immobilon P PVDF membrane, Immobilon Western ECL substrate solution (Millipore); Immobiline DryStrips, pH 3−10, 7 cm, and IPG buffer, pH 3−10 (GE Healthcare), were purchased from the manufacturers indicated in parentheses. .. Preparation of IPG Strips, pH 4−8, pH 4−10, and pH 4−11, 7 cm IPG strips were cast on GelBond PAG Film by the Bio-RAD model 475 delivery system for creating the gradient.

Derivative Assay:

Article Title: Conserved amino acid networks modulate discrete functional properties in an enzyme superfamily
Article Snippet: DNA constructs, expression and purification DNA sequences of RNase A and human RNase 3 (Eosinophil Cationic Protein) were Escherichia coli codon-optimized, and subcloned into Nde I/Hind III-digested expression vector pET22b(+) (EMD Biosciences, San Diego, CA, USA). .. DNA sequences of human RNase 2 (Eosinophil Derived-Neurotoxin), RNase 4 and RNase 5 (Angiogenin) were acquired from UniProt, Escherichia coli codon-optimized, and subcloned into Nde I/Hind III-digested expression vector pJexpress411/414 (DNA2.0, Menlo Park, CA, USA).

Article Title: Determinants of R-loop formation at convergent bidirectionally transcribed trinucleotide repeats
Article Snippet: Plasmids were subsequently treated with RNase A and T1 (Sigma), phenol extracted and purified twice by cesium chloride/ethidium bromide centrifugation and stored in TE (10 mM Tris, 1 mM EDTA pH 7.6) at −20°C. .. Plasmids containing (CGG) · (CCG) and (CAG) · (CTG) repeats were derived from FRAXA, SCA1 and DM1 patients, respectively, and were cloned into pPCRscript-AMP, pBluescript KS(+) or pGEM3Zf(+) ( ).

High Performance Liquid Chromatography:

Article Title: A Hinge Region Cis-proline in Ribonuclease A Acts as a Conformational Gatekeeper for C-terminal Domain Swapping
Article Snippet: Paragraph title: Evaluation of dimerization by HPLC Size Exclusion Chromatography ... The column was calibrated using a gel filtration molecular weight standard from Biorad and purified RNase A from Sigma.

Article Title: Modeling formalin fixation and antigen retrieval with bovine pancreatic ribonuclease A: I--Structural and functional alterations
Article Snippet: RNase A, 4 mg/ml, was incubated in neutral 10% formalin for 24 h, freed of formaldehyde by dialysis, and concentrated four-fold on a Microcon YM-10 centrifugal filter device (Millipore, Bedford, MA, USA). .. The column was eluted with 0.15 M KCl, 50 mM phosphate buffer (pH 7.4) with the use of a model 715 HPLC system (Gilson Medical Electronics, Middleton, WI, USA).

Flow Cytometry:

Article Title: Adsorption-Induced Changes in Ribonuclease A Structure and Enzymatic Activity on Solid Surfaces
Article Snippet: II.b Protein Adsorption and Equilibration The adsorption of RNAse A (Sigma R6513) on the material surfaces was carried out using previously described methods (see S.1.a in the ). .. Protein adsorption was conducted in 10 mM PPB under protein concentrations of 0.03 and 1.00 mg/mL for 2 h in order to vary the surface coverage of adsorbed protein on each surface, following which the material surfaces were gently rinsed under a steady gentle flow (12 mL/min) of PPB for 5 min to remove weakly adsorbed protein.

Sonication:

Article Title: Analysis of the oligomeric states of nucleophosmin using size exclusion chromatography
Article Snippet: The suspension was sonicated using a Handy Sonic (TOMY SEICO, Tokyo, Japan) at a power control setting of 5 for 20 sec. After centrifugation at 20,000 × g for 5 min at 4 °C, the supernatant was used as the ‘Triton X-100 insoluble fraction’. .. For RNase A treatment, cell lysates were treated with 40 µg/ml RNase A (Sigma-Aldrich) for 15 min at 37 °C.

Injection:

Article Title: MicroRNA Drop in the Bloodstream and MicroRNA Boost in the Tumour Caused by Treatment with Ribonuclease A Leads to an Attenuation of Tumour Malignancy
Article Snippet: LLC in solid form was induced by intramuscular (i.m.) injection of tumour cells (106 ) suspended in 0.1 ml of saline buffer into the right thighs of mice. .. On day 4 after tumour transplantation, mice were treated with saline buffer (n = 30) or RNase A (Sigma, USA) at a dose of 0.7 µg/kg (n = 20).

Article Title: Modeling formalin fixation and antigen retrieval with bovine pancreatic ribonuclease A: I--Structural and functional alterations
Article Snippet: RNase A, 4 mg/ml, was incubated in neutral 10% formalin for 24 h, freed of formaldehyde by dialysis, and concentrated four-fold on a Microcon YM-10 centrifugal filter device (Millipore, Bedford, MA, USA). .. The sample was then injected into an HR 16/50 column filled with prep grade Superdex 75 (Pharmacia Biotech, Uppsala, Sweden).

Article Title: Maternal RNA regulates Aurora C kinase during mouse oocyte maturation in a translation-independent fashion †
Article Snippet: The collection and injection medium for oocytes was bicarbonate-free minimal essential medium (MEM) containing 25 mM Hepes (pH 7.3), 3 mg/ml polyvinylpyrollidone (MEM/PVP), and 2.5 μM milrinone (Sigma #M4659) to prevent nuclear envelope breakdown and meiotic resumption [ ]. .. Each denuded oocyte was microinjected with ∼10 pl of 10, 50, or 100 μg/μl of RNase A (Sigma, R4642).

Molecular Weight:

Article Title: A Hinge Region Cis-proline in Ribonuclease A Acts as a Conformational Gatekeeper for C-terminal Domain Swapping
Article Snippet: .. The column was calibrated using a gel filtration molecular weight standard from Biorad and purified RNase A from Sigma. ..

Article Title: Electrolytic Reduction: Modification of Proteins Occurring in Isoelectric Focusing Electrophoresis and in Electrolytic Reactions in the Presence of High Salts
Article Snippet: .. Acrylamido buffers, 0.2 M stock solution (Fluka); GelBond PAG film (BioWhittaker); ribonuclease A, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), bovine insulin, and ovalbumin (Sigma); phenol red (Merck); low-ranged molecular weight markers (Calbiochem); EZ-Link Pentylamine-Biotin, EZ-Link biotin-LC-hydrazide, GelCode blue dye solution, and horseradish peroxidase-conjugated streptavidin (Thermo Scientific); OxyBlot Protein Oxidation Detection Kit (Chemicon); Immobilon P PVDF membrane, Immobilon Western ECL substrate solution (Millipore); Immobiline DryStrips, pH 3−10, 7 cm, and IPG buffer, pH 3−10 (GE Healthcare), were purchased from the manufacturers indicated in parentheses. .. Preparation of IPG Strips, pH 4−8, pH 4−10, and pH 4−11, 7 cm IPG strips were cast on GelBond PAG Film by the Bio-RAD model 475 delivery system for creating the gradient.

MTT Assay:

Article Title: Electrolytic Reduction: Modification of Proteins Occurring in Isoelectric Focusing Electrophoresis and in Electrolytic Reactions in the Presence of High Salts
Article Snippet: .. Acrylamido buffers, 0.2 M stock solution (Fluka); GelBond PAG film (BioWhittaker); ribonuclease A, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), bovine insulin, and ovalbumin (Sigma); phenol red (Merck); low-ranged molecular weight markers (Calbiochem); EZ-Link Pentylamine-Biotin, EZ-Link biotin-LC-hydrazide, GelCode blue dye solution, and horseradish peroxidase-conjugated streptavidin (Thermo Scientific); OxyBlot Protein Oxidation Detection Kit (Chemicon); Immobilon P PVDF membrane, Immobilon Western ECL substrate solution (Millipore); Immobiline DryStrips, pH 3−10, 7 cm, and IPG buffer, pH 3−10 (GE Healthcare), were purchased from the manufacturers indicated in parentheses. .. Preparation of IPG Strips, pH 4−8, pH 4−10, and pH 4−11, 7 cm IPG strips were cast on GelBond PAG Film by the Bio-RAD model 475 delivery system for creating the gradient.

Size-exclusion Chromatography:

Article Title: A Hinge Region Cis-proline in Ribonuclease A Acts as a Conformational Gatekeeper for C-terminal Domain Swapping
Article Snippet: Paragraph title: Evaluation of dimerization by HPLC Size Exclusion Chromatography ... The column was calibrated using a gel filtration molecular weight standard from Biorad and purified RNase A from Sigma.

Article Title: Analysis of the oligomeric states of nucleophosmin using size exclusion chromatography
Article Snippet: The suspension was sonicated using a Handy Sonic (TOMY SEICO, Tokyo, Japan) at a power control setting of 5 for 20 sec. After centrifugation at 20,000 × g for 5 min at 4 °C, the supernatant was used as the ‘Triton X-100 insoluble fraction’. .. For RNase A treatment, cell lysates were treated with 40 µg/ml RNase A (Sigma-Aldrich) for 15 min at 37 °C.

Mouse Assay:

Article Title: MicroRNA Drop in the Bloodstream and MicroRNA Boost in the Tumour Caused by Treatment with Ribonuclease A Leads to an Attenuation of Tumour Malignancy
Article Snippet: .. On day 4 after tumour transplantation, mice were treated with saline buffer (n = 30) or RNase A (Sigma, USA) at a dose of 0.7 µg/kg (n = 20). ..

Article Title: Maternal RNA regulates Aurora C kinase during mouse oocyte maturation in a translation-independent fashion †
Article Snippet: Prophase I-arrested oocytes were obtained from 6-week-old CF-1 female mice previously primed (44–48 h) with pregnant mare serum gonadotropin (PMSG) (Calbiochem #367 222) and mechanically stripped of cumulus cells [ ]. .. Each denuded oocyte was microinjected with ∼10 pl of 10, 50, or 100 μg/μl of RNase A (Sigma, R4642).

Purification:

Article Title: A Hinge Region Cis-proline in Ribonuclease A Acts as a Conformational Gatekeeper for C-terminal Domain Swapping
Article Snippet: .. The column was calibrated using a gel filtration molecular weight standard from Biorad and purified RNase A from Sigma. ..

Article Title: Nickel affects xylem Sap RNase a and converts RNase A to a urease
Article Snippet: Purified protein was denatured in SDS-gel sample buffer and electrophoresed on a SDS-10 to 20% polyacrylamide gradient gel. .. The nuclease activity of RNase A from bovine pancreas was verified by suspending RNA (ribonucleic acid from baker’s yeast, Saccharomyces cereviae; Sigma, St. Louis, Mo, USA) in Buffer E [25 mM 2-morpholineethanesulfonic acid (Mes), pH 6.2, 2.5 mM MnSO4 , and 2.5 mM DTT) [ ].

Article Title: Characterization of Intact Neo-Glycoproteins by Hydrophilic Interaction Liquid Chromatography
Article Snippet: .. Reagents and Chemicals α-Chymotrypsin, dithiothreitol (DTT), RNase A and RNase B from bovine pancreas were purchased from Sigma-Aldrich (Milan, Italy) and were used without further purification. .. Potassium dihydrogen phosphate and ammonium acetate were from Merck (Darmstadt, Germany) and of analytical grade purity.

Article Title: Conserved amino acid networks modulate discrete functional properties in an enzyme superfamily
Article Snippet: .. DNA constructs, expression and purification DNA sequences of RNase A and human RNase 3 (Eosinophil Cationic Protein) were Escherichia coli codon-optimized, and subcloned into Nde I/Hind III-digested expression vector pET22b(+) (EMD Biosciences, San Diego, CA, USA). .. DNA sequences of human RNase 2 (Eosinophil Derived-Neurotoxin), RNase 4 and RNase 5 (Angiogenin) were acquired from UniProt, Escherichia coli codon-optimized, and subcloned into Nde I/Hind III-digested expression vector pJexpress411/414 (DNA2.0, Menlo Park, CA, USA).

Article Title: Determinants of R-loop formation at convergent bidirectionally transcribed trinucleotide repeats
Article Snippet: .. Plasmids were subsequently treated with RNase A and T1 (Sigma), phenol extracted and purified twice by cesium chloride/ethidium bromide centrifugation and stored in TE (10 mM Tris, 1 mM EDTA pH 7.6) at −20°C. .. Plasmids containing (CGG) · (CCG) and (CAG) · (CTG) repeats were derived from FRAXA, SCA1 and DM1 patients, respectively, and were cloned into pPCRscript-AMP, pBluescript KS(+) or pGEM3Zf(+) ( ).

Plasmid Preparation:

Article Title: Conserved amino acid networks modulate discrete functional properties in an enzyme superfamily
Article Snippet: .. DNA constructs, expression and purification DNA sequences of RNase A and human RNase 3 (Eosinophil Cationic Protein) were Escherichia coli codon-optimized, and subcloned into Nde I/Hind III-digested expression vector pET22b(+) (EMD Biosciences, San Diego, CA, USA). .. DNA sequences of human RNase 2 (Eosinophil Derived-Neurotoxin), RNase 4 and RNase 5 (Angiogenin) were acquired from UniProt, Escherichia coli codon-optimized, and subcloned into Nde I/Hind III-digested expression vector pJexpress411/414 (DNA2.0, Menlo Park, CA, USA).

Article Title: Determinants of R-loop formation at convergent bidirectionally transcribed trinucleotide repeats
Article Snippet: Plasmids were subsequently treated with RNase A and T1 (Sigma), phenol extracted and purified twice by cesium chloride/ethidium bromide centrifugation and stored in TE (10 mM Tris, 1 mM EDTA pH 7.6) at −20°C. .. Plasmids containing (GAA) · (TTC) repeats were derived from the pGEM3Zf(−) plasmid, and were kindly provided by Robert D. Wells.

Spectrophotometric Assay:

Article Title: Adsorption-Induced Changes in Ribonuclease A Structure and Enzymatic Activity on Solid Surfaces
Article Snippet: II.e Characterization of Enzymatic Activity A spectrophotometric assay was used to measure the enzymatic activity of RNase A to complement the CD and AAL/MS data. .. Briefly, ribonucleic acid, which is the substrate for RNase A, was prepared in PPB to a final concentration of 20 mg/mL (Baker’s yeast, Sigma R6750) and exposed to RNase A both in solution and following RNase A adsorption.

In Vitro:

Article Title: Maternal RNA regulates Aurora C kinase during mouse oocyte maturation in a translation-independent fashion †
Article Snippet: Each denuded oocyte was microinjected with ∼10 pl of 10, 50, or 100 μg/μl of RNase A (Sigma, R4642). .. The microinjected oocytes were then incubated in Chatot, Ziomek, and Bavister (CZB) medium containing 2.5 μM milrinone for 1–2 h followed by in vitro maturation in milrinone-free CZB medium for 6 h (Met I) or 16 h (Met II) at 37°C in a humidified atmosphere of 5% CO2 in air.

Column Chromatography:

Article Title: Modeling formalin fixation and antigen retrieval with bovine pancreatic ribonuclease A: I--Structural and functional alterations
Article Snippet: Paragraph title: Column Chromatography ... RNase A, 4 mg/ml, was incubated in neutral 10% formalin for 24 h, freed of formaldehyde by dialysis, and concentrated four-fold on a Microcon YM-10 centrifugal filter device (Millipore, Bedford, MA, USA).

Incubation:

Article Title: A Hinge Region Cis-proline in Ribonuclease A Acts as a Conformational Gatekeeper for C-terminal Domain Swapping
Article Snippet: To initialize dimerization, purified protein was concentrated to 10 mg/mL in 100 mM Tris pH 8.0 and incubated at either 4°C, 25°C, or 37°C for periods of time ranging from 24 hours to 30 days. .. The column was calibrated using a gel filtration molecular weight standard from Biorad and purified RNase A from Sigma.

Article Title: Nickel affects xylem Sap RNase a and converts RNase A to a urease
Article Snippet: The nuclease activity of RNase A from bovine pancreas was verified by suspending RNA (ribonucleic acid from baker’s yeast, Saccharomyces cereviae; Sigma, St. Louis, Mo, USA) in Buffer E [25 mM 2-morpholineethanesulfonic acid (Mes), pH 6.2, 2.5 mM MnSO4 , and 2.5 mM DTT) [ ]. .. RNase A (10 μL) was then added to the RNA suspension and the reaction mixture (2 mg/ml) incubated at 25°C for 1 h and then centrifuged (5,500g at 4°C).

Article Title: Modeling formalin fixation and antigen retrieval with bovine pancreatic ribonuclease A: I--Structural and functional alterations
Article Snippet: .. RNase A, 4 mg/ml, was incubated in neutral 10% formalin for 24 h, freed of formaldehyde by dialysis, and concentrated four-fold on a Microcon YM-10 centrifugal filter device (Millipore, Bedford, MA, USA). .. The sample was then injected into an HR 16/50 column filled with prep grade Superdex 75 (Pharmacia Biotech, Uppsala, Sweden).

Article Title: Maternal RNA regulates Aurora C kinase during mouse oocyte maturation in a translation-independent fashion †
Article Snippet: Each denuded oocyte was microinjected with ∼10 pl of 10, 50, or 100 μg/μl of RNase A (Sigma, R4642). .. The microinjected oocytes were then incubated in Chatot, Ziomek, and Bavister (CZB) medium containing 2.5 μM milrinone for 1–2 h followed by in vitro maturation in milrinone-free CZB medium for 6 h (Met I) or 16 h (Met II) at 37°C in a humidified atmosphere of 5% CO2 in air.

Article Title: Analysis of the oligomeric states of nucleophosmin using size exclusion chromatography
Article Snippet: After incubation on ice for 10 min, samples were centrifuged at 2,100 × g for 5 min at 4 °C, then the supernatant was used as the ‘Triton X-100 soluble fraction’. .. For RNase A treatment, cell lysates were treated with 40 µg/ml RNase A (Sigma-Aldrich) for 15 min at 37 °C.

Concentration Assay:

Article Title: Adsorption-Induced Changes in Ribonuclease A Structure and Enzymatic Activity on Solid Surfaces
Article Snippet: II.b Protein Adsorption and Equilibration The adsorption of RNAse A (Sigma R6513) on the material surfaces was carried out using previously described methods (see S.1.a in the ). .. Briefly, 10 mM potassium phosphate buffer solution (PPB; pH 7.4) was prepared by mixing appropriate amounts of 1 M monobasic potassium phosphate (Sigma, P8708) or 1 M dibasic potassium phosphate (Sigma, P8508), following which the buffer concentration was verified by titrating against 0.065 M potassium hydrogen phthalate.

Article Title: Picornavirus RNA is protected from cleavage by ribonuclease during virion uncoating and transfer across cellular and model membranes
Article Snippet: .. Receptor-decorated liposomes were diluted to 2 mg/ml in the same rehydration buffer (described above) with RNase A (Sigma) added (except when noted) to a final concentration of 50 μg/ml. .. Covalent attachment of ribonuclease and fluorophores to virus Conjugation reactions (20 μl) were set up with PV (1 μg,) and RNase A (0, 7.5, 25 and 50 μg/ml, corresponding to approx.

Article Title: A phosphate-binding subsite in bovine pancreatic ribonuclease A can be converted into a very efficient catalytic site
Article Snippet: The kinetics of the hydrolysis reaction of the substrate cytidine 2′,3′-cyclic phosphate (C > p) (Sigma) by RNase A and variants were analyzed by the spectrophotometric method based on the absorbance increase of the reaction mixture at 296 nm due to the formation of 3′-CMP (Δɛ296 = 516.4 M−1 cm−1 ) ( ). .. The substrate concentration range was from 0.05 mM to 3 mM and the measurements were carried out at 25°C using 1-cm path-length cells.

Article Title: Maternal RNA regulates Aurora C kinase during mouse oocyte maturation in a translation-independent fashion †
Article Snippet: Each denuded oocyte was microinjected with ∼10 pl of 10, 50, or 100 μg/μl of RNase A (Sigma, R4642). .. Cycloheximide (Sigma, C7698) was dissolved in phosphate-buffered saline (PBS) and then added to CZB culture medium to a final concentration of 10 μg/ml.

Article Title: Adsorption-Induced Changes in Ribonuclease A Structure and Enzymatic Activity on Solid Surfaces
Article Snippet: .. Briefly, ribonucleic acid, which is the substrate for RNase A, was prepared in PPB to a final concentration of 20 mg/mL (Baker’s yeast, Sigma R6750) and exposed to RNase A both in solution and following RNase A adsorption. .. An initial calibration plot for solution-state enzymatic activity was obtained for a working range of 0.1–30 μg of RNAse A (based on the equivalently adsorbed amount of protein on different surfaces) by monitoring the absorbance at 300 nm (ΔA 300 ) at pH 7.4.

Fractionation:

Article Title: Analysis of the oligomeric states of nucleophosmin using size exclusion chromatography
Article Snippet: For fractionation of HL60 and OCI-AML3 cells, cells were lysed with PBS containing 1% Triton X-100, 15 mM imidazole, 1 mM DTT and 40 U/ml RNase inhibitor (Takara Bio). .. For RNase A treatment, cell lysates were treated with 40 µg/ml RNase A (Sigma-Aldrich) for 15 min at 37 °C.

CTG Assay:

Article Title: Determinants of R-loop formation at convergent bidirectionally transcribed trinucleotide repeats
Article Snippet: Plasmids were subsequently treated with RNase A and T1 (Sigma), phenol extracted and purified twice by cesium chloride/ethidium bromide centrifugation and stored in TE (10 mM Tris, 1 mM EDTA pH 7.6) at −20°C. .. Plasmids containing (CGG) · (CCG) and (CAG) · (CTG) repeats were derived from FRAXA, SCA1 and DM1 patients, respectively, and were cloned into pPCRscript-AMP, pBluescript KS(+) or pGEM3Zf(+) ( ).

Lysis:

Article Title: Analysis of the oligomeric states of nucleophosmin using size exclusion chromatography
Article Snippet: The resulting pellet was rinsed with lysis buffer and resuspended in new lysis buffer. .. For RNase A treatment, cell lysates were treated with 40 µg/ml RNase A (Sigma-Aldrich) for 15 min at 37 °C.

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  • 90
    Millipore rnase free milliq water
    Rnase Free Milliq Water, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rnase free milliq water/product/Millipore
    Average 90 stars, based on 2 article reviews
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    rnase free milliq water - by Bioz Stars, 2020-01
    90/100 stars
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    90
    Millipore anti rac1 antibody
    Dab2 and PAR-3 control VEGF receptor internalisation a , Alexa546-labelled VEGF-A or VEGF-C (red) accumulated in the perinuclear region of control mouse ECs at 30 min after stimulation, which was strongly reduced after knockdown of Dab2 or Pard3 . Actin, Phalloidin (green); nuclei, DAPI (blue). b, c , Quantitation of Alexa546-positive peri-nuclear VEGF-A ( b ) or VEGF-C ( c ) spots. Two different siRNAs were used for Dab2 and Pard3 in ( b ). Data represent the means±s.d. of 6 independent experiments. P values, two-tailed Student’s t-test. At least 100 cells were scored in each experiment. d, e , Biochemical detection of biotinylated (surface) VEGFR2 and VEGFR3 in stimulated control and Dab2 ( d ) or Pard3 ( e ) KD cells. Antibodies used for immunoblotting and molecular weight marker are indicated. f , Activation of <t>Rac1</t> in control and Dab2 or Pard3 KD mouse ECs stimulated with VEGF-A or VEGF-C for 5 min, as indicated.
    Anti Rac1 Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti rac1 antibody/product/Millipore
    Average 90 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    anti rac1 antibody - by Bioz Stars, 2020-01
    90/100 stars
      Buy from Supplier

    N/A
    Ribonucleases do not hydrolyze DNA because the DNA lacks 2 OH groups essential for the formation of cyclic intermediates RNase can hydrolyze RNA from protein samples Pancreatic RNase A specifically
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    Dab2 and PAR-3 control VEGF receptor internalisation a , Alexa546-labelled VEGF-A or VEGF-C (red) accumulated in the perinuclear region of control mouse ECs at 30 min after stimulation, which was strongly reduced after knockdown of Dab2 or Pard3 . Actin, Phalloidin (green); nuclei, DAPI (blue). b, c , Quantitation of Alexa546-positive peri-nuclear VEGF-A ( b ) or VEGF-C ( c ) spots. Two different siRNAs were used for Dab2 and Pard3 in ( b ). Data represent the means±s.d. of 6 independent experiments. P values, two-tailed Student’s t-test. At least 100 cells were scored in each experiment. d, e , Biochemical detection of biotinylated (surface) VEGFR2 and VEGFR3 in stimulated control and Dab2 ( d ) or Pard3 ( e ) KD cells. Antibodies used for immunoblotting and molecular weight marker are indicated. f , Activation of Rac1 in control and Dab2 or Pard3 KD mouse ECs stimulated with VEGF-A or VEGF-C for 5 min, as indicated.

    Journal: Nature cell biology

    Article Title: Spatial regulation of VEGF receptor endocytosis in angiogenesis

    doi: 10.1038/ncb2679

    Figure Lengend Snippet: Dab2 and PAR-3 control VEGF receptor internalisation a , Alexa546-labelled VEGF-A or VEGF-C (red) accumulated in the perinuclear region of control mouse ECs at 30 min after stimulation, which was strongly reduced after knockdown of Dab2 or Pard3 . Actin, Phalloidin (green); nuclei, DAPI (blue). b, c , Quantitation of Alexa546-positive peri-nuclear VEGF-A ( b ) or VEGF-C ( c ) spots. Two different siRNAs were used for Dab2 and Pard3 in ( b ). Data represent the means±s.d. of 6 independent experiments. P values, two-tailed Student’s t-test. At least 100 cells were scored in each experiment. d, e , Biochemical detection of biotinylated (surface) VEGFR2 and VEGFR3 in stimulated control and Dab2 ( d ) or Pard3 ( e ) KD cells. Antibodies used for immunoblotting and molecular weight marker are indicated. f , Activation of Rac1 in control and Dab2 or Pard3 KD mouse ECs stimulated with VEGF-A or VEGF-C for 5 min, as indicated.

    Article Snippet: Finally, 20 µl of each eluate was subjected to SDS-PAGE, followed by immunoblotting with anti-Rac1 antibody (Millipore, 05-389, 1:400).

    Techniques: Quantitation Assay, Two Tailed Test, Molecular Weight, Marker, Activation Assay