Structured Review

Millipore rnase a
The binding isotherm of <t>RNase</t> 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.
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Images

1) 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

2) Product Images from "Triple-Helical DNA in Drosophila Heterochromatin"

Article Title: Triple-Helical DNA in Drosophila Heterochromatin

Journal: Cells

doi: 10.3390/cells7120227

Polytene chromosome spreads of  D. melanogaster  wild type were treated with RNase A/RNase H mixture followed by proteinase K digestion in a time course experiment and subsequent immunological detection of triple-stranded DNA. DAPI staining (blue signal) and antibody labelling (red signal) were superimposed. Scale bar represents 25 µm.
Figure Legend Snippet: Polytene chromosome spreads of D. melanogaster wild type were treated with RNase A/RNase H mixture followed by proteinase K digestion in a time course experiment and subsequent immunological detection of triple-stranded DNA. DAPI staining (blue signal) and antibody labelling (red signal) were superimposed. Scale bar represents 25 µm.

Techniques Used: Staining

3) Product Images from "DIRS retrotransposons amplify via linear, single-stranded cDNA intermediates"

Article Title: DIRS retrotransposons amplify via linear, single-stranded cDNA intermediates

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkaa160

Features of extrachromosomal DIRS-1 bsr  cDNA. Extrachromosomal cDNA samples were extracted from rrpC– strains transformed with DIRS-1 bsr  ( A ) or DIRS-1 bsr*  ( B ), and were cultivated in G418/BS10 medium that selects for strains with mobilized retrotransposon. The samples were treated separately with RNase A, DNase I, Exonuclease I (Exo I), Exonuclease III (Exo III) and S1 nuclease (S1). After digestion and heat inactivation of enzymes, the samples were analyzed by PCR with the primers #2212 and #2213 binding within the region of  mbsrI  cassette (binding position in Figure   2A ). Quantification of endogenous ( C  and  D ) and  mbsrI -tagged versions ( E ) of DIRS-1 extrachromosomal cDNA after treatment with Exonuclease I and S1 nuclease, relative to the non-digested sample. Samples were extracted from rrpC– strains transformed with DIRS-1 bsr  and DIRS-1 bsr*  upon cultivation in G418/BS10 medium that selects for strains with mobilized retrotransposons. The cDNA abundance was monitored by qPCR using primers targeting positions qP1, qP2 and qBS (Figures   2A  and   3A ) and is shown in logarithmic scale relative to the non-digested sample (set to 1). Each qPCR reaction was run in triplicate.  Error bars : mean with S.D. Statistics: paired  t  test:  P
Figure Legend Snippet: Features of extrachromosomal DIRS-1 bsr cDNA. Extrachromosomal cDNA samples were extracted from rrpC– strains transformed with DIRS-1 bsr ( A ) or DIRS-1 bsr* ( B ), and were cultivated in G418/BS10 medium that selects for strains with mobilized retrotransposon. The samples were treated separately with RNase A, DNase I, Exonuclease I (Exo I), Exonuclease III (Exo III) and S1 nuclease (S1). After digestion and heat inactivation of enzymes, the samples were analyzed by PCR with the primers #2212 and #2213 binding within the region of mbsrI cassette (binding position in Figure 2A ). Quantification of endogenous ( C and D ) and mbsrI -tagged versions ( E ) of DIRS-1 extrachromosomal cDNA after treatment with Exonuclease I and S1 nuclease, relative to the non-digested sample. Samples were extracted from rrpC– strains transformed with DIRS-1 bsr and DIRS-1 bsr* upon cultivation in G418/BS10 medium that selects for strains with mobilized retrotransposons. The cDNA abundance was monitored by qPCR using primers targeting positions qP1, qP2 and qBS (Figures 2A and  3A ) and is shown in logarithmic scale relative to the non-digested sample (set to 1). Each qPCR reaction was run in triplicate. Error bars : mean with S.D. Statistics: paired t test: P

Techniques Used: Transformation Assay, Polymerase Chain Reaction, Binding Assay, Real-time Polymerase Chain Reaction

4) Product Images from "Human Nup98 regulates the localization and activity of DExH/D-box helicase DHX9"

Article Title: Human Nup98 regulates the localization and activity of DExH/D-box helicase DHX9

Journal: eLife

doi: 10.7554/eLife.18825

In vitro interaction of Nup98 and DHX9. ( A ) Anti-DHX9 antibodies coupled to beads were used to immobilize recombinant DHX9. Bead-bound DHX9 was then incubated with recombinant Nup98 or GST in the presence or absence of RNA (poly I:C), RNase A, or buffer alone. Bound proteins were analyzed by Western blots using the indicated antibodies (below images). The top row of panels shows the DHX9 bait bound to beads. The bottom row of panels shows GST and Nup98 that bound to DHX9 under the indicated conditions. Asterisks denote positions of DHX9 and Nup98. The positions of molecular mass markers (shown in kDa) are indicated on the left and right. ( B ) Example images of bead-bound complexes used for the quantification shown in   Figure 6C . ( C ) Example images of bead-bound complexes used for the quantification shown in   Figure 6D . DOI: http://dx.doi.org/10.7554/eLife.18825.014
Figure Legend Snippet: In vitro interaction of Nup98 and DHX9. ( A ) Anti-DHX9 antibodies coupled to beads were used to immobilize recombinant DHX9. Bead-bound DHX9 was then incubated with recombinant Nup98 or GST in the presence or absence of RNA (poly I:C), RNase A, or buffer alone. Bound proteins were analyzed by Western blots using the indicated antibodies (below images). The top row of panels shows the DHX9 bait bound to beads. The bottom row of panels shows GST and Nup98 that bound to DHX9 under the indicated conditions. Asterisks denote positions of DHX9 and Nup98. The positions of molecular mass markers (shown in kDa) are indicated on the left and right. ( B ) Example images of bead-bound complexes used for the quantification shown in Figure 6C . ( C ) Example images of bead-bound complexes used for the quantification shown in Figure 6D . DOI: http://dx.doi.org/10.7554/eLife.18825.014

Techniques Used: In Vitro, Recombinant, Incubation, Western Blot

5) Product Images from "Antagonism of the Protein Kinase R Pathway in Human Cells by Rhesus Cytomegalovirus"

Article Title: Antagonism of the Protein Kinase R Pathway in Human Cells by Rhesus Cytomegalovirus

Journal: Journal of Virology

doi: 10.1128/JVI.01793-17

RhCMV generates more dsRNA than HCMV does in HF. (A) Production of dsRNA in HF infected with RhCMV and HCMV. HF were mock infected or infected with RhCMV and HCMV (MOI = 3), and at the indicated times postinfection, whole-cell RNA was harvested; 200 ng of mock-infected or infected cell RNA or the indicated amounts of reovirus RNA were blotted onto nitrocellulose and analyzed using antiserum that recognizes dsRNA (9D5 antibody) as described in Materials and Methods. Samples from 48 h postinfection and reovirus RNA were also treated with RNase T1 and RNase A prior to blotting. (B) The amounts of dsRNA generated by RhCMV and HCMV during three separate infections were quantified as described above, and the average quantities from these replicates with standard deviations are shown. The amounts of dsRNA present at each time point were not significantly different from each other, with the exception of dsRNA from mock-infected cells at 48 h compared to RhCMV dsRNA from the same time point ( P = 0.0475; unpaired t test). Mock-infected cell dsRNA was not collected at 72 h.
Figure Legend Snippet: RhCMV generates more dsRNA than HCMV does in HF. (A) Production of dsRNA in HF infected with RhCMV and HCMV. HF were mock infected or infected with RhCMV and HCMV (MOI = 3), and at the indicated times postinfection, whole-cell RNA was harvested; 200 ng of mock-infected or infected cell RNA or the indicated amounts of reovirus RNA were blotted onto nitrocellulose and analyzed using antiserum that recognizes dsRNA (9D5 antibody) as described in Materials and Methods. Samples from 48 h postinfection and reovirus RNA were also treated with RNase T1 and RNase A prior to blotting. (B) The amounts of dsRNA generated by RhCMV and HCMV during three separate infections were quantified as described above, and the average quantities from these replicates with standard deviations are shown. The amounts of dsRNA present at each time point were not significantly different from each other, with the exception of dsRNA from mock-infected cells at 48 h compared to RhCMV dsRNA from the same time point ( P = 0.0475; unpaired t test). Mock-infected cell dsRNA was not collected at 72 h.

Techniques Used: Infection, Generated

6) Product Images from "The molecular mechanism for activating IgA production by Pediococcus acidilactici K15 and the clinical impact in a randomized trial"

Article Title: The molecular mechanism for activating IgA production by Pediococcus acidilactici K15 and the clinical impact in a randomized trial

Journal: Scientific Reports

doi: 10.1038/s41598-018-23404-4

IL-6 and IL-10 production by mDC1s in response to LAB ( a ) mDC1s were isolated from PBMCs of 7 donors. mDC1s were stimulated with heat-killed LAB in duplicates for 24 h. The tested strains LAB and their abbreviations are described in Table S1 . The resulting IL-6 and IL-10 concentrations were measured by ELISA. Data are represented as mean ± SD of 7 donors. ( b ) Heat-killed K15 cells were treated with RNase A under 0 M NaCl for digestion of ssRNA and dsRNA or under 0.3 M NaCl for digestion of ssRNA only. mDC1s were cultured with heat-killed K15 or RNase A-treated, heat-killed K15 for 24 h. The resulting IL-6 and IL-10 concentrations were measured by ELISA. Data are represented as the mean ± SD of duplicates and are representative of two independent experiments from different individuals. * p
Figure Legend Snippet: IL-6 and IL-10 production by mDC1s in response to LAB ( a ) mDC1s were isolated from PBMCs of 7 donors. mDC1s were stimulated with heat-killed LAB in duplicates for 24 h. The tested strains LAB and their abbreviations are described in Table S1 . The resulting IL-6 and IL-10 concentrations were measured by ELISA. Data are represented as mean ± SD of 7 donors. ( b ) Heat-killed K15 cells were treated with RNase A under 0 M NaCl for digestion of ssRNA and dsRNA or under 0.3 M NaCl for digestion of ssRNA only. mDC1s were cultured with heat-killed K15 or RNase A-treated, heat-killed K15 for 24 h. The resulting IL-6 and IL-10 concentrations were measured by ELISA. Data are represented as the mean ± SD of duplicates and are representative of two independent experiments from different individuals. * p

Techniques Used: Isolation, Enzyme-linked Immunosorbent Assay, Cell Culture

7) Product Images from "Post-translational modification directs nuclear and hyphal tip localization of C. albicans mRNA-binding protein Slr1"

Article Title: Post-translational modification directs nuclear and hyphal tip localization of C. albicans mRNA-binding protein Slr1

Journal: Molecular microbiology

doi: 10.1111/mmi.13643

Slr1-GFP co-fractionates with 80S and translating ribosomes A. Log-phase yeast cells expressing Slr1-GFP ( SLR1-GFP/slr1 Δ) were treated with cycloheximide prior to lysis. Lysates were loaded on linear 7–47% sucrose gradients and fractionated following centrifugation. RNA absorbance at 254 nm was measured during fractionation to detect 40S and 60S ribosomal subunits, 80S ribosomes and polysomes. B. TCA-precipitated fractions were resolved by SDS-10% PAGE and tested for the presence of Slr1-GFP and ribosomal protein Rps3 by immunoblot (GFP, uS3). The percent of each protein present in non-ribosomal and ribosomal fractions was determined using Image StudioLite software (LI-COR). C. Lysates were treated with RNase A prior to sucrose-density gradient centrifugation and then analyzed as in (B).
Figure Legend Snippet: Slr1-GFP co-fractionates with 80S and translating ribosomes A. Log-phase yeast cells expressing Slr1-GFP ( SLR1-GFP/slr1 Δ) were treated with cycloheximide prior to lysis. Lysates were loaded on linear 7–47% sucrose gradients and fractionated following centrifugation. RNA absorbance at 254 nm was measured during fractionation to detect 40S and 60S ribosomal subunits, 80S ribosomes and polysomes. B. TCA-precipitated fractions were resolved by SDS-10% PAGE and tested for the presence of Slr1-GFP and ribosomal protein Rps3 by immunoblot (GFP, uS3). The percent of each protein present in non-ribosomal and ribosomal fractions was determined using Image StudioLite software (LI-COR). C. Lysates were treated with RNase A prior to sucrose-density gradient centrifugation and then analyzed as in (B).

Techniques Used: Expressing, Lysis, Centrifugation, Fractionation, Polyacrylamide Gel Electrophoresis, Software, Gradient Centrifugation

8) Product Images from "Highly selective toxic and proapoptotic effects of two dimeric ribonucleases on thyroid cancer cells compared to the effects of doxorubicin"

Article Title: Highly selective toxic and proapoptotic effects of two dimeric ribonucleases on thyroid cancer cells compared to the effects of doxorubicin

Journal: British Journal of Cancer

doi: 10.1038/sj.bjc.6601491

Time-dependent effect of BS-RNase (•-•) and HHP2-RNase (▪-▪) on tumour growth in mice inoculated s.c. (day 0) with 1 × 10 6 ARO cells. RNases were administered in the peritumoral area six times at 72 h intervals as indicated by the arrows. Controls were treated with PBS (□-□) or RNase A (○-○). Latency periods and means of the weights of the tumours excised at the end of each treatment are reported in the table inset.
Figure Legend Snippet: Time-dependent effect of BS-RNase (•-•) and HHP2-RNase (▪-▪) on tumour growth in mice inoculated s.c. (day 0) with 1 × 10 6 ARO cells. RNases were administered in the peritumoral area six times at 72 h intervals as indicated by the arrows. Controls were treated with PBS (□-□) or RNase A (○-○). Latency periods and means of the weights of the tumours excised at the end of each treatment are reported in the table inset.

Techniques Used: Mouse Assay

9) Product Images from "Mutations in domain a? of protein disulfide isomerase affect the folding pathway of bovine pancreatic ribonuclease A"

Article Title: Mutations in domain a? of protein disulfide isomerase affect the folding pathway of bovine pancreatic ribonuclease A

Journal: Protein Science : A Publication of the Protein Society

doi:

( A ). ( B ) Early stages of the time-course shown in A . ( C ) Time-course analysis of folding in the presence of wild-type protein disulfide isomerase (PDI). PDI was incubated with 1.5 mM GSH/0.3 mM GSSG for 10 min at 25°C and then added to RNase A (1 mg/mL = 73 μM) at a concentration of 10 μM. Percentages of intermediates were derived by electrospray ionization mass spectrometry analysis. The differences between folding experiments performed completely independent of each other were about 5%. For the sake of clarity, error bars are not shown. n S represents intramolecular disulfide bonds, n G mixed disulfides with glutathione, and n H free thiols.
Figure Legend Snippet: ( A ). ( B ) Early stages of the time-course shown in A . ( C ) Time-course analysis of folding in the presence of wild-type protein disulfide isomerase (PDI). PDI was incubated with 1.5 mM GSH/0.3 mM GSSG for 10 min at 25°C and then added to RNase A (1 mg/mL = 73 μM) at a concentration of 10 μM. Percentages of intermediates were derived by electrospray ionization mass spectrometry analysis. The differences between folding experiments performed completely independent of each other were about 5%. For the sake of clarity, error bars are not shown. n S represents intramolecular disulfide bonds, n G mixed disulfides with glutathione, and n H free thiols.

Techniques Used: Incubation, Concentration Assay, Derivative Assay, Mass Spectrometry

Appearance of the 4S species in the RNase A folding, as detected by electrospray ionization mass spectrometry (ESIMS) analysis under different conditions, relative to the recovery of enzyme activity from aliquots of the same folding reactions. The continuous and dotted lines refer to the activity data and ESIMS data, respectively.
Figure Legend Snippet: Appearance of the 4S species in the RNase A folding, as detected by electrospray ionization mass spectrometry (ESIMS) analysis under different conditions, relative to the recovery of enzyme activity from aliquots of the same folding reactions. The continuous and dotted lines refer to the activity data and ESIMS data, respectively.

Techniques Used: Mass Spectrometry, Activity Assay

10) Product Images from "Centromere Chromosome Orientation Fluorescent in situ Hybridization (Cen-CO-FISH) Detects Sister Chromatid Exchange at the Centromere in Human Cells"

Article Title: Centromere Chromosome Orientation Fluorescent in situ Hybridization (Cen-CO-FISH) Detects Sister Chromatid Exchange at the Centromere in Human Cells

Journal: Bio-protocol

doi: 10.21769/BioProtoc.2792

Representative images of the Cen-CO-FISH procedure at different stages of the protocol. A. Cells are harvested, hypotonically swollen and fixed overnight. Samples can be stored at 4 °C at this stage. Cells are then dropped onto glass slides to let the metaphases spread. Slides can be stored in the dark at room temperature (RT) before proceeding to Step B. B. Rehydrate the slides into PBS in a Coplin jar for 5 min. C. Remove PBS and add RNase A solution into the jar, incubate in a 37 °C water bath for 10 min. Remove RNase solution and incubate with Hoechst in 2x SSC for 15 min at RT. D. Place slides into plastic try and cover with just enough 2x SSC. E. Place tray into Stratalinker oven with 365 nm UV bulbs and expose for 30 min (1,800 sec). F. Prepare 80 μl of Exonuclease III solution for each slide and add the solution to a 24 × 60 coverslip that will make contact with the entire surface of the slide. G. Immediately after UV exposure, pick up the coverslip with Exonuclease III solution as shown. Invert the slide, adjust the coverslip to make sure it is central, the liquid is well distributed and there are no air bubbles. Incubate cells side up for 10 min at RT and then repeat Steps F-G one more time for an additional Exonuclease incubation. Wash in PBS and dehydrate in ethanol series. Store slides at RT in the dark (overnight). H. Prepare one of the probes (1:1,000–1:5,000) into hybridization solution, heat for 10 min at 60 °C before adding 80 μl onto the coverslips. Proceed as in Steps F-G shown. I. Place slides into a Hybridization chamber and incubate for 2 h at RT. To make a chamber, take a box, fill it with paper towels and wet throughout until all towels are humid. Do not put the slides directly on top of the wet paper. Use pipettes or other forms of support to raise the coverslips over the wet paper. Place slides cells side up and close the box away from light. Upon opening the box after 2 h, you should see a small amount of condensation on the lid. Wash with Hybridization buffer #1 and repeat Step H with the second, reverse complement probe (you can invert the order of the hybridization between forward and reverse complement probes, it should yield identical results). Wash in Hybridization buffer #1 and #2, including the DAPI step, dehydrate and air dry for at least one hour. Mount and seal before imaging.
Figure Legend Snippet: Representative images of the Cen-CO-FISH procedure at different stages of the protocol. A. Cells are harvested, hypotonically swollen and fixed overnight. Samples can be stored at 4 °C at this stage. Cells are then dropped onto glass slides to let the metaphases spread. Slides can be stored in the dark at room temperature (RT) before proceeding to Step B. B. Rehydrate the slides into PBS in a Coplin jar for 5 min. C. Remove PBS and add RNase A solution into the jar, incubate in a 37 °C water bath for 10 min. Remove RNase solution and incubate with Hoechst in 2x SSC for 15 min at RT. D. Place slides into plastic try and cover with just enough 2x SSC. E. Place tray into Stratalinker oven with 365 nm UV bulbs and expose for 30 min (1,800 sec). F. Prepare 80 μl of Exonuclease III solution for each slide and add the solution to a 24 × 60 coverslip that will make contact with the entire surface of the slide. G. Immediately after UV exposure, pick up the coverslip with Exonuclease III solution as shown. Invert the slide, adjust the coverslip to make sure it is central, the liquid is well distributed and there are no air bubbles. Incubate cells side up for 10 min at RT and then repeat Steps F-G one more time for an additional Exonuclease incubation. Wash in PBS and dehydrate in ethanol series. Store slides at RT in the dark (overnight). H. Prepare one of the probes (1:1,000–1:5,000) into hybridization solution, heat for 10 min at 60 °C before adding 80 μl onto the coverslips. Proceed as in Steps F-G shown. I. Place slides into a Hybridization chamber and incubate for 2 h at RT. To make a chamber, take a box, fill it with paper towels and wet throughout until all towels are humid. Do not put the slides directly on top of the wet paper. Use pipettes or other forms of support to raise the coverslips over the wet paper. Place slides cells side up and close the box away from light. Upon opening the box after 2 h, you should see a small amount of condensation on the lid. Wash with Hybridization buffer #1 and repeat Step H with the second, reverse complement probe (you can invert the order of the hybridization between forward and reverse complement probes, it should yield identical results). Wash in Hybridization buffer #1 and #2, including the DAPI step, dehydrate and air dry for at least one hour. Mount and seal before imaging.

Techniques Used: Fluorescence In Situ Hybridization, Size-exclusion Chromatography, Incubation, Hybridization, Imaging

11) 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

12) 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

13) 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

14) Product Images from "A bacterial group II intron encoding reverse transcriptase, maturase, and DNA endonuclease activities: biochemical demonstration of maturase activity and insertion of new genetic information within the intron"

Article Title: A bacterial group II intron encoding reverse transcriptase, maturase, and DNA endonuclease activities: biochemical demonstration of maturase activity and insertion of new genetic information within the intron

Journal: Genes & Development

doi:

Reverse splicing of the Ll.ltrB intron into the E1E2 DNA substrate. ( A ) Reverse splicing reactions with internally labeled DNA substrate. RNP particles (0.025 OD 260 unit) from cells grown at 37°C were incubated with the 129-bp 32 P-labeled E1E2 DNA substrate (150,000 cpm, ∼125 fmoles). The products were denatured with glyoxal and analyzed in a 1% agarose gel. (Lane 1 ) DNA substrate incubated in the absence of RNP particles; (lane 2 ) DNA substrate incubated with RNP particles from cells expressing pLI1; (lanes 3–6 ) reverse spliced products of pLI1 RNP particles treated with RNase A, alkali, S1 nuclease, or DNase I; (lanes 7,8 ) pLI1 RNP particles pretreated with RNase A or proteinase K prior to reverse splicing (see Materials and Methods); (lanes 9–11 ) DNA substrate incubated with RNP particles from cells containing pET-11a, pLI1–FS, and pLI1P, respectively. ( B ) Reverse splicing reaction with E1E2 DNA substrates labeled separately at each of the four termini. pLI1 RNP particles were incubated with 5′- or 3′-end-labeled DNA substrates (150,000 cpm; ∼250 fmoles of 5′-end-labeled substrates and ∼200 fmoles of 3′-end-labeled substrates), and the products were analyzed as above. Numbers to the left indicate DNA size markers (kb).
Figure Legend Snippet: Reverse splicing of the Ll.ltrB intron into the E1E2 DNA substrate. ( A ) Reverse splicing reactions with internally labeled DNA substrate. RNP particles (0.025 OD 260 unit) from cells grown at 37°C were incubated with the 129-bp 32 P-labeled E1E2 DNA substrate (150,000 cpm, ∼125 fmoles). The products were denatured with glyoxal and analyzed in a 1% agarose gel. (Lane 1 ) DNA substrate incubated in the absence of RNP particles; (lane 2 ) DNA substrate incubated with RNP particles from cells expressing pLI1; (lanes 3–6 ) reverse spliced products of pLI1 RNP particles treated with RNase A, alkali, S1 nuclease, or DNase I; (lanes 7,8 ) pLI1 RNP particles pretreated with RNase A or proteinase K prior to reverse splicing (see Materials and Methods); (lanes 9–11 ) DNA substrate incubated with RNP particles from cells containing pET-11a, pLI1–FS, and pLI1P, respectively. ( B ) Reverse splicing reaction with E1E2 DNA substrates labeled separately at each of the four termini. pLI1 RNP particles were incubated with 5′- or 3′-end-labeled DNA substrates (150,000 cpm; ∼250 fmoles of 5′-end-labeled substrates and ∼200 fmoles of 3′-end-labeled substrates), and the products were analyzed as above. Numbers to the left indicate DNA size markers (kb).

Techniques Used: Labeling, Incubation, Agarose Gel Electrophoresis, Expressing, Positron Emission Tomography

15) Product Images from "Pressure versus temperature unfolding of ribonuclease A: An FTIR spectroscopic characterization of 10 variants at the carboxy-terminal site"

Article Title: Pressure versus temperature unfolding of ribonuclease A: An FTIR spectroscopic characterization of 10 variants at the carboxy-terminal site

Journal: Protein Science : A Publication of the Protein Society

doi:

( A ) Original and ( B ) Fourier-deconvoluted and fitted IR spectra in the amide I′ region (solid line) with individual Gaussian components (broken lines) of RNase A wild type; ( C ) normalised Fourier-deconvoluted IR spectra of RNase A wild type (solid line) and V108G variant (broken line). Solution conditions: RNase A and V108G at 75 mg/mL in sodium acetate buffer, 50 mM, pD 5.0, P = 0.1 MPa, T = 12°C.
Figure Legend Snippet: ( A ) Original and ( B ) Fourier-deconvoluted and fitted IR spectra in the amide I′ region (solid line) with individual Gaussian components (broken lines) of RNase A wild type; ( C ) normalised Fourier-deconvoluted IR spectra of RNase A wild type (solid line) and V108G variant (broken line). Solution conditions: RNase A and V108G at 75 mg/mL in sodium acetate buffer, 50 mM, pD 5.0, P = 0.1 MPa, T = 12°C.

Techniques Used: Variant Assay

Normalised Fourier deconvoluted IR spectra of the pressure (1200 MPa, 20°C) ( A ) and temperature (0.1 MPa, 85°C) ( B ). Denatured RNase A wild type (solid line) and V108G variant (broken line).
Figure Legend Snippet: Normalised Fourier deconvoluted IR spectra of the pressure (1200 MPa, 20°C) ( A ) and temperature (0.1 MPa, 85°C) ( B ). Denatured RNase A wild type (solid line) and V108G variant (broken line).

Techniques Used: Variant Assay

16) Product Images from "Tomato Ringspot Virus Proteins Containing the Nucleoside Triphosphate Binding Domain Are Transmembrane Proteins That Associate with the Endoplasmic Reticulum and Cofractionate with Replication Complexes †"

Article Title: Tomato Ringspot Virus Proteins Containing the Nucleoside Triphosphate Binding Domain Are Transmembrane Proteins That Associate with the Endoplasmic Reticulum and Cofractionate with Replication Complexes †

Journal: Journal of Virology

doi: 10.1128/JVI.77.1.523-534.2003

Analysis of membrane-bound ToRSV RdRp activity. (A) Detection of [α- 32 P]UTP-labeled RdRp products synthesized with P30-2 fractions from ToRSV-infected (lanes I) and healthy (lanes H) plants. The RdRp products were separated on 1% agarose gels. Total products (lanes 1 and 2), DNase I- and actinomycin D (Act.D)-resistant products (lanes 3 and 4), and RNase A-resistant products (lanes 5 and 6) are shown. The migrations of ToRSV ssRNA1 and ssRNA2 (ss1 and ss2, respectively) and dsRNAs purified from infected plants (the double-stranded forms of RNA1 and RNA2 do not separate on 1% agarose gels and migrate as one diffuse band) are shown on the left. In lane 5, the presence of 2% SDS in the proteinase K digestion buffer used after the RNase A treatment interfered slightly with the migration of the sample, resulting in a slower migration of the dsRNA products. (B) Dot blot hybridization analysis of labeled RdRp products with P30-2 fractions from ToRSV-infected (lanes I) and healthy (lanes H) plants. Transcripts corresponding to the positive (+) or negative (−) strand of a region of ToRSV RNA2 were synthesized in vitro as described in Materials and Methods and blotted onto Zeta-Probe membranes (Bio-Rad). The polarity of the ToRSV transcripts is indicated at the top. (C) Fractionation of RdRp activity, proteins containing the NTB or VPg domain, Bip, and rRNAs in a 20 to 45% sucrose gradient with P30-2 extracts from ToRSV-infected tissue. Each fraction was analyzed for RNase A-resistant RdRp activity (top); subjected to immunoblot analysis using anti-NTB, anti-VPg, and anti-Bip antibodies; and tested for the presence of rRNA. For the anti-NTB and anti-VPg antibodies, only the portion of the gel containing the predominant 66/69-kDa band is shown.
Figure Legend Snippet: Analysis of membrane-bound ToRSV RdRp activity. (A) Detection of [α- 32 P]UTP-labeled RdRp products synthesized with P30-2 fractions from ToRSV-infected (lanes I) and healthy (lanes H) plants. The RdRp products were separated on 1% agarose gels. Total products (lanes 1 and 2), DNase I- and actinomycin D (Act.D)-resistant products (lanes 3 and 4), and RNase A-resistant products (lanes 5 and 6) are shown. The migrations of ToRSV ssRNA1 and ssRNA2 (ss1 and ss2, respectively) and dsRNAs purified from infected plants (the double-stranded forms of RNA1 and RNA2 do not separate on 1% agarose gels and migrate as one diffuse band) are shown on the left. In lane 5, the presence of 2% SDS in the proteinase K digestion buffer used after the RNase A treatment interfered slightly with the migration of the sample, resulting in a slower migration of the dsRNA products. (B) Dot blot hybridization analysis of labeled RdRp products with P30-2 fractions from ToRSV-infected (lanes I) and healthy (lanes H) plants. Transcripts corresponding to the positive (+) or negative (−) strand of a region of ToRSV RNA2 were synthesized in vitro as described in Materials and Methods and blotted onto Zeta-Probe membranes (Bio-Rad). The polarity of the ToRSV transcripts is indicated at the top. (C) Fractionation of RdRp activity, proteins containing the NTB or VPg domain, Bip, and rRNAs in a 20 to 45% sucrose gradient with P30-2 extracts from ToRSV-infected tissue. Each fraction was analyzed for RNase A-resistant RdRp activity (top); subjected to immunoblot analysis using anti-NTB, anti-VPg, and anti-Bip antibodies; and tested for the presence of rRNA. For the anti-NTB and anti-VPg antibodies, only the portion of the gel containing the predominant 66/69-kDa band is shown.

Techniques Used: Activity Assay, Labeling, Synthesized, Infection, Activated Clotting Time Assay, Purification, Migration, Dot Blot, Hybridization, In Vitro, Fractionation

17) Product Images from "Triple-Helical DNA in Drosophila Heterochromatin"

Article Title: Triple-Helical DNA in Drosophila Heterochromatin

Journal: Cells

doi: 10.3390/cells7120227

Polytene chromosome spreads of  D. melanogaster  wild type were treated with RNase A/RNase H mixture followed by proteinase K digestion in a time course experiment and subsequent immunological detection of triple-stranded DNA. DAPI staining (blue signal) and antibody labelling (red signal) were superimposed. Scale bar represents 25 µm.
Figure Legend Snippet: Polytene chromosome spreads of D. melanogaster wild type were treated with RNase A/RNase H mixture followed by proteinase K digestion in a time course experiment and subsequent immunological detection of triple-stranded DNA. DAPI staining (blue signal) and antibody labelling (red signal) were superimposed. Scale bar represents 25 µm.

Techniques Used: Staining

18) Product Images from "FrnE, a Cadmium-Inducible Protein in Deinococcus radiodurans, Is Characterized as a Disulfide Isomerase Chaperone In Vitro and for Its Role in Oxidative Stress Tolerance In Vivo"

Article Title: FrnE, a Cadmium-Inducible Protein in Deinococcus radiodurans, Is Characterized as a Disulfide Isomerase Chaperone In Vitro and for Its Role in Oxidative Stress Tolerance In Vivo

Journal: Journal of Bacteriology

doi: 10.1128/JB.01503-12

In vitro activity characterization of recombinant drFrnE. (A) In brief, 40 μM scrambled RNase A was incubated with 10 μM purified recombinant drFrnE (+FrnE) for disulfide isomerase activity assay as described in Materials and Methods,
Figure Legend Snippet: In vitro activity characterization of recombinant drFrnE. (A) In brief, 40 μM scrambled RNase A was incubated with 10 μM purified recombinant drFrnE (+FrnE) for disulfide isomerase activity assay as described in Materials and Methods,

Techniques Used: In Vitro, Activity Assay, Recombinant, Incubation, Purification

19) Product Images from "Dynamic interactions within sub-complexes of the H/ACA pseudouridylation guide RNP"

Article Title: Dynamic interactions within sub-complexes of the H/ACA pseudouridylation guide RNP

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkm673

Single-stranded nuclease footprinting of Pf9. ( A ) 5′-end labelled Pf9 was digested with indicated concentrations of RNase A (lanes 3, 4) or RNase T1 (lanes 5, 6). The cleavage products were separated on a denaturing 20% acrylamide gel. Lane 1 is undigested RNA and lane 2 is a size marker generated by alkaline hydrolysis (OH). Strong cleavages at nucleotides in the upper stem region are indicated with red arrowheads. ( B ) Summary of Pf9 RNA cleavages by single-stranded nucleases in the context of the predicted secondary structure of Pf9 RNA (as in Figure 1 ). Upper stem region is boxed in red.
Figure Legend Snippet: Single-stranded nuclease footprinting of Pf9. ( A ) 5′-end labelled Pf9 was digested with indicated concentrations of RNase A (lanes 3, 4) or RNase T1 (lanes 5, 6). The cleavage products were separated on a denaturing 20% acrylamide gel. Lane 1 is undigested RNA and lane 2 is a size marker generated by alkaline hydrolysis (OH). Strong cleavages at nucleotides in the upper stem region are indicated with red arrowheads. ( B ) Summary of Pf9 RNA cleavages by single-stranded nucleases in the context of the predicted secondary structure of Pf9 RNA (as in Figure 1 ). Upper stem region is boxed in red.

Techniques Used: Footprinting, Acrylamide Gel Assay, Marker, Generated

Single-stranded nuclease footprinting of L7Ae–Pf9. ( A ) 5′-end labelled Pf9 was incubated alone (lanes 3, 5) or with 1 μM L7Ae (lanes 4, 6) and digested with RNase T1 (lanes 3, 4) or RNase A (lanes 5, 6). The cleavage products were separated on a denaturing 15% acrylamide gel. Lane 1 is undigested RNA and lane 2 is a size marker generated by alkaline hydrolysis (OH). Red arrowheads indicate cleavages in the upper stem of the guide RNA in the absence of protein. Green and yellow bars indicate strong L7Ae protections and cleavage enhancements, respectively. ( B ) Summary of L7Ae cleavage protections and enhancements in the context of the predicted secondary structure of Pf9 (as in Figure 1 ).
Figure Legend Snippet: Single-stranded nuclease footprinting of L7Ae–Pf9. ( A ) 5′-end labelled Pf9 was incubated alone (lanes 3, 5) or with 1 μM L7Ae (lanes 4, 6) and digested with RNase T1 (lanes 3, 4) or RNase A (lanes 5, 6). The cleavage products were separated on a denaturing 15% acrylamide gel. Lane 1 is undigested RNA and lane 2 is a size marker generated by alkaline hydrolysis (OH). Red arrowheads indicate cleavages in the upper stem of the guide RNA in the absence of protein. Green and yellow bars indicate strong L7Ae protections and cleavage enhancements, respectively. ( B ) Summary of L7Ae cleavage protections and enhancements in the context of the predicted secondary structure of Pf9 (as in Figure 1 ).

Techniques Used: Footprinting, Incubation, Acrylamide Gel Assay, Marker, Generated

20) Product Images from "Mutagenic Analysis of the 3? cis-Acting Elements of the Rubella Virus Genome"

Article Title: Mutagenic Analysis of the 3? cis-Acting Elements of the Rubella Virus Genome

Journal: Journal of Virology

doi:

Analysis of SL2/SL3 conformations by RNase probing. RNA probes consisting of the 3′-terminal 90 nt plus a poly(A) tract from the fTH or HPV77 (HPV) strain were digested with a battery of single- or double-stranded RNases, and the digestion pattern was resolved by primer extension. (A) Results of primer extension from pUC3′RUB110-fTH transcripts digested in a 20-μl reaction with no RNase (−; lanes 1 and 19); mung bean nuclease (MB; cleaves single-stranded RNA with no nucleotide specificity), 10 (lane 2) and 5 (lane 3) U; RNase T 2 (cleaves single-stranded RNA with no nucleotide specificity), 0.5 (lane 4), 0.25 (lane 5), 0.1 (lane 6), and 0.05 (lane 7) U; RNase V 1 (cleaves double-stranded RNA with no nucleotide specificity), 0.1 (lane 12), 0.05 (lane 13), and 0.025 (lane 14) U; and RNase A (cleaves single-stranded RNA with preference for C and U), 0.1 (lane 20), 0.05 (lane 21), 0.025 (lane 22), and 0.01 (lane 23) U. In lanes 8 to 11 and 15 to 18 are the sequencing ladders for orientation produced by using the primer extension primer and plasmid pUCRUB3′110-fTH as a template. Structural regions within the probe are shown on both margins. Digestion landmarks highlighted include the loops of SL2 and SL3 ( > ), both of which are sensitive to the single-stranded RNases but not RNase V 1 (the SL3 loop is not sensitive to RNase A because it contains a GAAA sequence), and the single efficient RNase V 1 digestion site in SL2 and the digestion of the 5′ side of the SL4 stem by RNase V 1 (≫). The overall results of RNase probing on both probes are summarized in panels B (single-stranded RNases) and C (double-stranded RNase V 1 ). Because the primer was complementary to the 11 nt preceding the poly(A) tract (italics), digestion within these nucleotides could not be resolved. Single-stranded RNases: T1, RNase T 1 (G residue); T2, RNase T 2 (no specificity); A, RNase A (C and U residues); MB, mung bean nuclease (no specificity); U2, RNase U 2 (A residue).
Figure Legend Snippet: Analysis of SL2/SL3 conformations by RNase probing. RNA probes consisting of the 3′-terminal 90 nt plus a poly(A) tract from the fTH or HPV77 (HPV) strain were digested with a battery of single- or double-stranded RNases, and the digestion pattern was resolved by primer extension. (A) Results of primer extension from pUC3′RUB110-fTH transcripts digested in a 20-μl reaction with no RNase (−; lanes 1 and 19); mung bean nuclease (MB; cleaves single-stranded RNA with no nucleotide specificity), 10 (lane 2) and 5 (lane 3) U; RNase T 2 (cleaves single-stranded RNA with no nucleotide specificity), 0.5 (lane 4), 0.25 (lane 5), 0.1 (lane 6), and 0.05 (lane 7) U; RNase V 1 (cleaves double-stranded RNA with no nucleotide specificity), 0.1 (lane 12), 0.05 (lane 13), and 0.025 (lane 14) U; and RNase A (cleaves single-stranded RNA with preference for C and U), 0.1 (lane 20), 0.05 (lane 21), 0.025 (lane 22), and 0.01 (lane 23) U. In lanes 8 to 11 and 15 to 18 are the sequencing ladders for orientation produced by using the primer extension primer and plasmid pUCRUB3′110-fTH as a template. Structural regions within the probe are shown on both margins. Digestion landmarks highlighted include the loops of SL2 and SL3 ( > ), both of which are sensitive to the single-stranded RNases but not RNase V 1 (the SL3 loop is not sensitive to RNase A because it contains a GAAA sequence), and the single efficient RNase V 1 digestion site in SL2 and the digestion of the 5′ side of the SL4 stem by RNase V 1 (≫). The overall results of RNase probing on both probes are summarized in panels B (single-stranded RNases) and C (double-stranded RNase V 1 ). Because the primer was complementary to the 11 nt preceding the poly(A) tract (italics), digestion within these nucleotides could not be resolved. Single-stranded RNases: T1, RNase T 1 (G residue); T2, RNase T 2 (no specificity); A, RNase A (C and U residues); MB, mung bean nuclease (no specificity); U2, RNase U 2 (A residue).

Techniques Used: Sequencing, Produced, Plasmid Preparation

21) Product Images from "Reverse Transcriptase (RT) Inhibition of PCR at Low Concentrations of Template and Its Implications for Quantitative RT-PCR"

Article Title: Reverse Transcriptase (RT) Inhibition of PCR at Low Concentrations of Template and Its Implications for Quantitative RT-PCR

Journal: Applied and Environmental Microbiology

doi:

RT inhibition of RT-PCR demonstrated on Nostoc sp. nifH mRNA. Lanes: M, φX174 × Hae III molecular weight marker; 1, 5 μl of purified mRNA plus RT; 2, 5 μl of purified mRNA without RT; 3 and 4, same as 1 and 2, except that the mRNA sample was pretreated with RNase A prior to RT-PCR; 5, 20 pg of Nostoc sp. genomic DNA subject to PCR amplification only; 6, 2 pg of genomic DNA; 7, 200 fg of genomic DNA; 8, no-template control.
Figure Legend Snippet: RT inhibition of RT-PCR demonstrated on Nostoc sp. nifH mRNA. Lanes: M, φX174 × Hae III molecular weight marker; 1, 5 μl of purified mRNA plus RT; 2, 5 μl of purified mRNA without RT; 3 and 4, same as 1 and 2, except that the mRNA sample was pretreated with RNase A prior to RT-PCR; 5, 20 pg of Nostoc sp. genomic DNA subject to PCR amplification only; 6, 2 pg of genomic DNA; 7, 200 fg of genomic DNA; 8, no-template control.

Techniques Used: Inhibition, Reverse Transcription Polymerase Chain Reaction, Molecular Weight, Marker, Purification, Polymerase Chain Reaction, Amplification

RT-PCR with a dilution series of purified N. europaea 16S rRNA as template. +, RT present; −, RT absent. Lanes: M, φX174 × Hae III molecular weight marker; 1 and 2, 2 ng of 16S rRNA with or without RT; 3 and 4, 200 pg; 5 and 6, 20 pg; 7 and 8, 2 pg; 9 and 10, 200 fg; 11 and 12, 20 fg; 13 and 14, 2 fg; 15 and 16, no-template control; 17 and 18, 2 ng of N. europaea genomic DNA; 19 to 24, same as lanes 1 to 6, except that the sample was pretreated with RNase A prior to RT-PCR; 25 and 26, no-template controls pretreated with RNase A; 27 and 28, 2 ng of N. europaea genomic DNA pretreated with RNase A; 29, 20 pg of genomic DNA, PCR-only control; 30, 2 pg of genomic DNA; 31, 200 fg of genomic DNA; 32, no-template PCR-only control.
Figure Legend Snippet: RT-PCR with a dilution series of purified N. europaea 16S rRNA as template. +, RT present; −, RT absent. Lanes: M, φX174 × Hae III molecular weight marker; 1 and 2, 2 ng of 16S rRNA with or without RT; 3 and 4, 200 pg; 5 and 6, 20 pg; 7 and 8, 2 pg; 9 and 10, 200 fg; 11 and 12, 20 fg; 13 and 14, 2 fg; 15 and 16, no-template control; 17 and 18, 2 ng of N. europaea genomic DNA; 19 to 24, same as lanes 1 to 6, except that the sample was pretreated with RNase A prior to RT-PCR; 25 and 26, no-template controls pretreated with RNase A; 27 and 28, 2 ng of N. europaea genomic DNA pretreated with RNase A; 29, 20 pg of genomic DNA, PCR-only control; 30, 2 pg of genomic DNA; 31, 200 fg of genomic DNA; 32, no-template PCR-only control.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Purification, Molecular Weight, Marker, Polymerase Chain Reaction

22) Product Images from "A role for transportin in deposition of TTP to cytoplasmic RNA granules and mRNA decay"

Article Title: A role for transportin in deposition of TTP to cytoplasmic RNA granules and mRNA decay

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkp717

The interaction between TRN and TTP. HEK293 cells were transiently transfected with the FLAG-TTP expression vector or empty vector. ( A ) Cells were treated with arsenite or mock-treated for 1 h before harvest. The cell lysates were subjected to immunoprecipitation with anti-FLAG in the presence of RNase A. The precipitates were analyzed by immunoblotting with anti-TRN and anti-FLAG. ( B ) The  in vitro  translation reaction containing  35 S-labeled TTP was incubated with GST or GST-TRN followed by chromatography on glutathione-Sepharose. Bound proteins were fractionated on SDS–PAGE and detected by autoradiography. ( C ) HeLa cells that transiently expressed FLAG-TTP were double immunostained with anti-FLAG and anti-TRN. ( D ) Upper panel:  35 S-labeled HuR and TTP proteins were each subjected to the pull-down assay as in B. Recombinant RanQ69L was loaded with GTP and subsequently added into the pull-down reaction (lanes 4 and 8). Bottom panel: HeLa cells that transiently expressed FLAG-TTP and HA-Ran or HA-RanQ69L were double immunostained with anti-FLAG and anti-HA.
Figure Legend Snippet: The interaction between TRN and TTP. HEK293 cells were transiently transfected with the FLAG-TTP expression vector or empty vector. ( A ) Cells were treated with arsenite or mock-treated for 1 h before harvest. The cell lysates were subjected to immunoprecipitation with anti-FLAG in the presence of RNase A. The precipitates were analyzed by immunoblotting with anti-TRN and anti-FLAG. ( B ) The in vitro translation reaction containing 35 S-labeled TTP was incubated with GST or GST-TRN followed by chromatography on glutathione-Sepharose. Bound proteins were fractionated on SDS–PAGE and detected by autoradiography. ( C ) HeLa cells that transiently expressed FLAG-TTP were double immunostained with anti-FLAG and anti-TRN. ( D ) Upper panel: 35 S-labeled HuR and TTP proteins were each subjected to the pull-down assay as in B. Recombinant RanQ69L was loaded with GTP and subsequently added into the pull-down reaction (lanes 4 and 8). Bottom panel: HeLa cells that transiently expressed FLAG-TTP and HA-Ran or HA-RanQ69L were double immunostained with anti-FLAG and anti-HA.

Techniques Used: Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, In Vitro, Labeling, Incubation, Chromatography, SDS Page, Autoradiography, Pull Down Assay, Recombinant

23) Product Images from "HuR Displaces Polypyrimidine Tract Binding Protein To Facilitate La Binding to the 3′ Untranslated Region and Enhances Hepatitis C Virus Replication"

Article Title: HuR Displaces Polypyrimidine Tract Binding Protein To Facilitate La Binding to the 3′ Untranslated Region and Enhances Hepatitis C Virus Replication

Journal: Journal of Virology

doi: 10.1128/JVI.01714-15

Interactions between HuR and La at the 3′ UTR. (A) A GST pulldown assay was performed by incubating GST- or GST-HuR-coated beads with Huh7 or replicon cell lysates. The beads were either treated with RNase A or left untreated. The beads along
Figure Legend Snippet: Interactions between HuR and La at the 3′ UTR. (A) A GST pulldown assay was performed by incubating GST- or GST-HuR-coated beads with Huh7 or replicon cell lysates. The beads were either treated with RNase A or left untreated. The beads along

Techniques Used: GST Pulldown Assay

24) Product Images from "Effect of HM910, a novel camptothecin derivative, on the inhibition of multiple myeloma cell growth in vitro and in vivo"

Article Title: Effect of HM910, a novel camptothecin derivative, on the inhibition of multiple myeloma cell growth in vitro and in vivo

Journal: American Journal of Cancer Research

doi:

HM910 induced cell cycle arrest in G1 phase in MM cells. A. Cells were stained with PBS containing Triton X-100 (0.12%), EDTA (0.12 mmol/L), RNase A (100 µg/mL) and propidium iodide (50 µg/mL), and the cell cycle stages of MM cells were determined by flow cytometry. Representative results were exhibited, similar results were obtained from two other independent trials. B. Data shown as means ± SD, from triplicate independent determinations. C. HM910 or topotecan had effect on CDK2, CDK4, CDK6, cyclin D1 and c-myc protein levels determined by Western bloting. D. Gray value analysis of CDKs and C-myc determined by Image J. β-actin was used as a loading control. Data shown as means ± SD using triplicate independent determinations.
Figure Legend Snippet: HM910 induced cell cycle arrest in G1 phase in MM cells. A. Cells were stained with PBS containing Triton X-100 (0.12%), EDTA (0.12 mmol/L), RNase A (100 µg/mL) and propidium iodide (50 µg/mL), and the cell cycle stages of MM cells were determined by flow cytometry. Representative results were exhibited, similar results were obtained from two other independent trials. B. Data shown as means ± SD, from triplicate independent determinations. C. HM910 or topotecan had effect on CDK2, CDK4, CDK6, cyclin D1 and c-myc protein levels determined by Western bloting. D. Gray value analysis of CDKs and C-myc determined by Image J. β-actin was used as a loading control. Data shown as means ± SD using triplicate independent determinations.

Techniques Used: Staining, Flow Cytometry, Cytometry, Western Blot

25) Product Images from "Amyloids assemble as part of recognizable structures during oogenesis in Xenopus"

Article Title: Amyloids assemble as part of recognizable structures during oogenesis in Xenopus

Journal: Biology Open

doi: 10.1242/bio.017384

Maintenance of nucleolin and thio-T staining of isolated Xenopus nuclei (GVs) is RNA dependent. (A-C) Alexa Fluor 568 Phalloidin-stained (red) stage V-VI Xenopus GVs were left untreated (A) or treated with RNase A (B) or Xrn1 (C) for 30 min in the presence of 50 μM thio-T (green). Scale bars: 100 μm. (D-F) Untreated (D) or RNase A-treated (E) GVs were immunostained for nucleolin (magenta). (F) Dark field image nucleus. Scale bars: 100 μm.
Figure Legend Snippet: Maintenance of nucleolin and thio-T staining of isolated Xenopus nuclei (GVs) is RNA dependent. (A-C) Alexa Fluor 568 Phalloidin-stained (red) stage V-VI Xenopus GVs were left untreated (A) or treated with RNase A (B) or Xrn1 (C) for 30 min in the presence of 50 μM thio-T (green). Scale bars: 100 μm. (D-F) Untreated (D) or RNase A-treated (E) GVs were immunostained for nucleolin (magenta). (F) Dark field image nucleus. Scale bars: 100 μm.

Techniques Used: Staining, Isolation

26) Product Images from "Mass Spectrometric Evidence for the Existence of Distinct Modifications of Different Proteins by 2(E), 4(E)-Decadienal"

Article Title: Mass Spectrometric Evidence for the Existence of Distinct Modifications of Different Proteins by 2(E), 4(E)-Decadienal

Journal: Chemical research in toxicology

doi: 10.1021/tx900379a

MALDI-TOF mass spectra of 0.25 mM cytochrome c (A) or RNase A (B) modified by 20 equivalents of DDE (5 mM) in pH 7.4 phosphate buffer at 37 °C for various times. The mass increment of 134 Da is due to the DDE–Lys Schiff base formation.
Figure Legend Snippet: MALDI-TOF mass spectra of 0.25 mM cytochrome c (A) or RNase A (B) modified by 20 equivalents of DDE (5 mM) in pH 7.4 phosphate buffer at 37 °C for various times. The mass increment of 134 Da is due to the DDE–Lys Schiff base formation.

Techniques Used: Modification

27) 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

28) Product Images from "Engineered Picornavirus VPg-RNA Substrates: Analysis of a Tyrosyl-RNA Phosphodiesterase Activity"

Article Title: Engineered Picornavirus VPg-RNA Substrates: Analysis of a Tyrosyl-RNA Phosphodiesterase Activity

Journal: PLoS ONE

doi: 10.1371/journal.pone.0016559

Optimal assay conditions for detecting unlinkase activity using full-length poliovirus virion RNA 35 S-methionine labeled substrate. A protein chromatography fraction enriched for unlinkase activity was incubated with full-length 35 S-methionine radiolabeled W1-VPg 31 virion RNA substrate with either (A) increasing amounts of protein (0.01, 0.02, 0.04, 0.1, 0.2, 0.4, and 0.6 µg/µl) from an enriched source of unlinkase activity (Fraction SA) at 30°C for 30 minutes or (B) increasing incubation time (0, 1, 2, 5, 10, 15, 20, and 30 minutes) with 0.4 µg/µl of protein from a partially-purified fraction of unlinkase activity (Fraction SA) to determine optimal assay conditions for the full-length substrate. (C) 35 S-methionine radiolabeled W1-VPg 31 virion RNA substrate was mock-incubated (lane 1), incubated with 0.8 µg/µl RSW (lane 2), 0.4 µg/µl of protein from a partially-purified fraction of unlinkase activity (Fraction SA) (lane 3), or one unit of RNase A (lane 4) to differentiate between non-specific nuclease activity and authentic unlinkase activity.
Figure Legend Snippet: Optimal assay conditions for detecting unlinkase activity using full-length poliovirus virion RNA 35 S-methionine labeled substrate. A protein chromatography fraction enriched for unlinkase activity was incubated with full-length 35 S-methionine radiolabeled W1-VPg 31 virion RNA substrate with either (A) increasing amounts of protein (0.01, 0.02, 0.04, 0.1, 0.2, 0.4, and 0.6 µg/µl) from an enriched source of unlinkase activity (Fraction SA) at 30°C for 30 minutes or (B) increasing incubation time (0, 1, 2, 5, 10, 15, 20, and 30 minutes) with 0.4 µg/µl of protein from a partially-purified fraction of unlinkase activity (Fraction SA) to determine optimal assay conditions for the full-length substrate. (C) 35 S-methionine radiolabeled W1-VPg 31 virion RNA substrate was mock-incubated (lane 1), incubated with 0.8 µg/µl RSW (lane 2), 0.4 µg/µl of protein from a partially-purified fraction of unlinkase activity (Fraction SA) (lane 3), or one unit of RNase A (lane 4) to differentiate between non-specific nuclease activity and authentic unlinkase activity.

Techniques Used: Activity Assay, Labeling, Chromatography, Incubation, Purification

29) Product Images from "Promiscuous protein biotinylation by Escherichia coli biotin protein ligase"

Article Title: Promiscuous protein biotinylation by Escherichia coli biotin protein ligase

Journal: Protein Science : A Publication of the Protein Society

doi: 10.1110/ps.04911804

In vitro biotinylation with purified wild type and R118G proteins. In vitro biotinylation reactions were initiated by addition of purified wild type or R118G mutant BirA proteins (to a final concentration of 20 nM) to a reaction mixture containing a 2 μM final concentration of a specific substrate (apo BCCP) or a promiscuous acceptor (BSA or RNAse A) followed by incubation for 1 h at 37°C as described in Materials and Methods, followed by SDS-PAGE. Biotinylation of each protein was then analyzed by Western blotting with streptavidin-AP. (Lane 1 ) apo BCCP lacking BirA (note that this apo BCCP preparation was slightly contaminated with holo BCCP accounting for the bands in lanes 1 and 5 ); (lane 2 ) apo BCCP with wild type BirA; (lane 3 ) BSA with wild type BirA; (lane 4 ) RNAse A with wild type BirA; (lane 5 ) apo BCCP lacking R118G BirA; (lane 6 ) apo BCCP with R118G BirA; (lane 7 ) BSA with R118G BirA; (lane 8 ) RNAse A with R118G BirA. The arrows show the migration positions of BSA, BCCP, and RNase A.
Figure Legend Snippet: In vitro biotinylation with purified wild type and R118G proteins. In vitro biotinylation reactions were initiated by addition of purified wild type or R118G mutant BirA proteins (to a final concentration of 20 nM) to a reaction mixture containing a 2 μM final concentration of a specific substrate (apo BCCP) or a promiscuous acceptor (BSA or RNAse A) followed by incubation for 1 h at 37°C as described in Materials and Methods, followed by SDS-PAGE. Biotinylation of each protein was then analyzed by Western blotting with streptavidin-AP. (Lane 1 ) apo BCCP lacking BirA (note that this apo BCCP preparation was slightly contaminated with holo BCCP accounting for the bands in lanes 1 and 5 ); (lane 2 ) apo BCCP with wild type BirA; (lane 3 ) BSA with wild type BirA; (lane 4 ) RNAse A with wild type BirA; (lane 5 ) apo BCCP lacking R118G BirA; (lane 6 ) apo BCCP with R118G BirA; (lane 7 ) BSA with R118G BirA; (lane 8 ) RNAse A with R118G BirA. The arrows show the migration positions of BSA, BCCP, and RNase A.

Techniques Used: In Vitro, Purification, Mutagenesis, Concentration Assay, Incubation, SDS Page, Western Blot, Migration

30) Product Images from "U7 snRNP-specific Lsm11 protein: dual binding contacts with the 100 kDa zinc finger processing factor (ZFP100) and a ZFP100-independent function in histone RNA 3? end processing"

Article Title: U7 snRNP-specific Lsm11 protein: dual binding contacts with the 100 kDa zinc finger processing factor (ZFP100) and a ZFP100-independent function in histone RNA 3? end processing

Journal: Nucleic Acids Research

doi: 10.1093/nar/gki516

Determinants of ZFP100–U7 snRNP interactions in mammalian cell extracts. ( A ) The various HA-tagged proteins indicated on the left were expressed in human 293-T cells by transient transfection. Whole cell extracts were subjected to immunoprecipitation with affinity-purified antibodies directed against the N-terminal 169 amino acids of human ZFP100 (α-ZFP). The samples were subjected to SDS–PAGE, blotted, and the HA-tagged proteins were revealed by anti-HA antibody. For the two top panels, the extracts were incubated in the absence (lanes 1–3) or presence of RNase A (lanes 4–6) prior to immunoprecipitation (see Materials and Methods). The samples for each protein were processed in the same experiment and analysed on the same gel, but an intermediate lane was excised from the picture. FL, full-length murine Lsm11 (HA-mLsm11 FL ); ΔN140, mLsm11 lacking the first 140 amino acids (HA-mLsm11 Δ140 ); N136-D2, first 136 amino acids of mLsm11 fused to Sm D2 ( 16 ). Beads, precipitation by protein G sepharose beads without antibody; input, 1/20 of original extract. ( B ) Wild-type HA-tagged Lsm11 and the clustered point mutants indicated on the right (for their sequence see Figure 3 ) were expressed in human 293-T cells and their ability to interact with ZFP100 was assessed by immunoprecipitation with α-ZFP as in (A). ( C ) Nuclear extract from HeLa cells was subjected to precipitation with glutathione beads coupled to GST-ZFP 1–169 (lane 3) or GST alone (lane 2) and analysed for several snRNP components as indicated on the right. Lsm11 was detected by affinity-purified anti-Lsm11 antibodies ( 16 ); Sm B/B′ and D1 by the monoclonal anti-Sm antibody Y12; and snRNAs U1, U2 and U7 by RT–PCR as described in Materials and Methods. Input, 1/10 of original extract.
Figure Legend Snippet: Determinants of ZFP100–U7 snRNP interactions in mammalian cell extracts. ( A ) The various HA-tagged proteins indicated on the left were expressed in human 293-T cells by transient transfection. Whole cell extracts were subjected to immunoprecipitation with affinity-purified antibodies directed against the N-terminal 169 amino acids of human ZFP100 (α-ZFP). The samples were subjected to SDS–PAGE, blotted, and the HA-tagged proteins were revealed by anti-HA antibody. For the two top panels, the extracts were incubated in the absence (lanes 1–3) or presence of RNase A (lanes 4–6) prior to immunoprecipitation (see Materials and Methods). The samples for each protein were processed in the same experiment and analysed on the same gel, but an intermediate lane was excised from the picture. FL, full-length murine Lsm11 (HA-mLsm11 FL ); ΔN140, mLsm11 lacking the first 140 amino acids (HA-mLsm11 Δ140 ); N136-D2, first 136 amino acids of mLsm11 fused to Sm D2 ( 16 ). Beads, precipitation by protein G sepharose beads without antibody; input, 1/20 of original extract. ( B ) Wild-type HA-tagged Lsm11 and the clustered point mutants indicated on the right (for their sequence see Figure 3 ) were expressed in human 293-T cells and their ability to interact with ZFP100 was assessed by immunoprecipitation with α-ZFP as in (A). ( C ) Nuclear extract from HeLa cells was subjected to precipitation with glutathione beads coupled to GST-ZFP 1–169 (lane 3) or GST alone (lane 2) and analysed for several snRNP components as indicated on the right. Lsm11 was detected by affinity-purified anti-Lsm11 antibodies ( 16 ); Sm B/B′ and D1 by the monoclonal anti-Sm antibody Y12; and snRNAs U1, U2 and U7 by RT–PCR as described in Materials and Methods. Input, 1/10 of original extract.

Techniques Used: Transfection, Immunoprecipitation, Affinity Purification, SDS Page, Incubation, Sequencing, Reverse Transcription Polymerase Chain Reaction

31) Product Images from "A hepatitis C virus (HCV) internal ribosome entry site (IRES) domain III-IV-targeted aptamer inhibits translation by binding to an apical loop of domain IIId"

Article Title: A hepatitis C virus (HCV) internal ribosome entry site (IRES) domain III-IV-targeted aptamer inhibits translation by binding to an apical loop of domain IIId

Journal: Nucleic Acids Research

doi: 10.1093/nar/gki215

( A ) RNase A mapping of aptamer 3-07. 5′ end-labeled aptamer 3-07 was digested with RNase A. Left and right figures show 5′ and 3′side cleavage pattern of 3-07 aptamer, respectively. End-labeled aptamer was applied to a PAGE without further reactions (lanes 1 and 5). Alkaline ladder (lanes 2 and 6) and RNase U2 digestion (lanes 3 and 7) were used as markers. RNase U2 digestion carried out under the U2 buffer. Aptamer 3-07 was digested with RNase A (C and U specific cleavage) under the selection buffer (lanes 4 and 8). ( B ) Predicted secondary structure of aptamer 3-07. Arrowheads and their size indicate cleavage site by RNase A and cleavage intensity, respectively.
Figure Legend Snippet: ( A ) RNase A mapping of aptamer 3-07. 5′ end-labeled aptamer 3-07 was digested with RNase A. Left and right figures show 5′ and 3′side cleavage pattern of 3-07 aptamer, respectively. End-labeled aptamer was applied to a PAGE without further reactions (lanes 1 and 5). Alkaline ladder (lanes 2 and 6) and RNase U2 digestion (lanes 3 and 7) were used as markers. RNase U2 digestion carried out under the U2 buffer. Aptamer 3-07 was digested with RNase A (C and U specific cleavage) under the selection buffer (lanes 4 and 8). ( B ) Predicted secondary structure of aptamer 3-07. Arrowheads and their size indicate cleavage site by RNase A and cleavage intensity, respectively.

Techniques Used: Labeling, Polyacrylamide Gel Electrophoresis, Selection

32) Product Images from "mPEG-PAMAM-G4 Nucleic Acid Nanocomplexes: Enhanced Stability, RNase Protection, and Activity of Splice Switching Oligomer and Poly I:C RNA"

Article Title: mPEG-PAMAM-G4 Nucleic Acid Nanocomplexes: Enhanced Stability, RNase Protection, and Activity of Splice Switching Oligomer and Poly I:C RNA

Journal: Biomacromolecules

doi: 10.1021/bm4012425

RNA protection in fetal bovine serum and RNase A. mPEG-PAMAM-G4 was complexed with RNA at N/P ratios of 10 (Row A), 5 (Row B), 1 (Row C), 0.4 (Row D), and 0.2 (Row E). Lanes 1, 3, 5, 7, and 9 have mPEG-PAMAM-G4, and lanes 2, 4, 6, 8, and 10 lack mPEG-PAMAM-G4.
Figure Legend Snippet: RNA protection in fetal bovine serum and RNase A. mPEG-PAMAM-G4 was complexed with RNA at N/P ratios of 10 (Row A), 5 (Row B), 1 (Row C), 0.4 (Row D), and 0.2 (Row E). Lanes 1, 3, 5, 7, and 9 have mPEG-PAMAM-G4, and lanes 2, 4, 6, 8, and 10 lack mPEG-PAMAM-G4.

Techniques Used:

33) Product Images from "Transcriptional arrest: Escherichia coli RNA polymerase translocates backward, leaving the 3? end of the RNA intact and extruded"

Article Title: Transcriptional arrest: Escherichia coli RNA polymerase translocates backward, leaving the 3? end of the RNA intact and extruded

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

doi:

Effect of arrest on the transcript arrangement in RNAP. ( A ) RNase A footprinting of the transcript in active EC 26 and in arrested EC 27 . The RNA in the complexes was internally labeled at positions +26A or +12C. Each sample was treated with RNase A and fractionated by centrifugation into soluble (s) and matrix-associated (p) fractions before gel analysis. Sequences of the transcripts are shown alongside the autoradiograms, asterisks mark positions of labeling, and arrows show major cuts introduced into the RNA (bold shaded line) by RNase A. The cylinders represent the transcript segments protected by RNAP in the active and arrested complexes. Nonfractionated samples (t) are included as controls. ( B ) 5′-Terminal phosphorylation of the transcripts. RNA-labeled EC 20 and EC 27 (lanes 6 and 4) and arrested fraction of EC 27 purified by chase (lane 2) were treated with T4 polynucleotide kinase in the presence of ATP (lanes 5, 3, and 1). The symbol (P) indicates the phosphorylated transcripts. Arrows indicate the mobility of phosphorylated and nonphosphorylated transcripts.
Figure Legend Snippet: Effect of arrest on the transcript arrangement in RNAP. ( A ) RNase A footprinting of the transcript in active EC 26 and in arrested EC 27 . The RNA in the complexes was internally labeled at positions +26A or +12C. Each sample was treated with RNase A and fractionated by centrifugation into soluble (s) and matrix-associated (p) fractions before gel analysis. Sequences of the transcripts are shown alongside the autoradiograms, asterisks mark positions of labeling, and arrows show major cuts introduced into the RNA (bold shaded line) by RNase A. The cylinders represent the transcript segments protected by RNAP in the active and arrested complexes. Nonfractionated samples (t) are included as controls. ( B ) 5′-Terminal phosphorylation of the transcripts. RNA-labeled EC 20 and EC 27 (lanes 6 and 4) and arrested fraction of EC 27 purified by chase (lane 2) were treated with T4 polynucleotide kinase in the presence of ATP (lanes 5, 3, and 1). The symbol (P) indicates the phosphorylated transcripts. Arrows indicate the mobility of phosphorylated and nonphosphorylated transcripts.

Techniques Used: Footprinting, Labeling, Centrifugation, Purification

34) Product Images from "Interaction of aurintricarboxylic acid (ATA) with four nucleic acid binding proteins DNase I, RNase A, reverse transcriptase and Taq polymerase"

Article Title: Interaction of aurintricarboxylic acid (ATA) with four nucleic acid binding proteins DNase I, RNase A, reverse transcriptase and Taq polymerase

Journal: Spectrochimica Acta. Part A, Molecular and Biomolecular Spectroscopy

doi: 10.1016/j.saa.2009.09.024

The near-UV and far-UV (inset) CD spectra of 3.52 μM of RNase A (solid line) and 9 μM ATA bound RNase A (dotted line) in RNase assay buffer was measured in Hitachi at 25 °C.
Figure Legend Snippet: The near-UV and far-UV (inset) CD spectra of 3.52 μM of RNase A (solid line) and 9 μM ATA bound RNase A (dotted line) in RNase assay buffer was measured in Hitachi at 25 °C.

Techniques Used:

Binding isotherm for the interaction of RNase A with ATA at 25 °C. The tryptophan fluorescence emission intensity ( λ ex = 295 nm, λ em = 340 nm) was plotted as a function of ligand (ATA) concentration C L . Inset shows the corresponding plot of 1/Δ F (Δ F denotes the change in fluorescence) against 1/( C L − C P ) to evaluate the dissociation constant by means of Eq. (1) , where C P is the protein concentration.
Figure Legend Snippet: Binding isotherm for the interaction of RNase A with ATA at 25 °C. The tryptophan fluorescence emission intensity ( λ ex = 295 nm, λ em = 340 nm) was plotted as a function of ligand (ATA) concentration C L . Inset shows the corresponding plot of 1/Δ F (Δ F denotes the change in fluorescence) against 1/( C L − C P ) to evaluate the dissociation constant by means of Eq. (1) , where C P is the protein concentration.

Techniques Used: Binding Assay, Fluorescence, Concentration Assay, Protein Concentration

35) Product Images from "BRD4 inhibitors block telomere elongation"

Article Title: BRD4 inhibitors block telomere elongation

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkx561

BRD4 inhibition does not affect telomerase enzyme activity. ( A ) Direct telomerase assay using whole cell lysates of 293TRex cells overexpressing hTR, TERT, POT1 and TPP1, which were treated with DMSO (lane 1), 0.5 μM JQ1 (lane 2), 2.5 μM OTX015 (lane 3), 25 μM MS436 (lane 4) or 5 μM IBET151 (lane 5). Lanes 6–10 show extracts pretreated with RNase A to show activity due to RNase sensitive telomerase enzyme. ( B ) Quantification of telomerase processivity and ( C ) Quantification of telomerase catalytic activity as described in ‘Materials and Methods’ section. Values in (B) and (C) are averages of two technical replicates. Error bars in (C) represent the standard deviation.
Figure Legend Snippet: BRD4 inhibition does not affect telomerase enzyme activity. ( A ) Direct telomerase assay using whole cell lysates of 293TRex cells overexpressing hTR, TERT, POT1 and TPP1, which were treated with DMSO (lane 1), 0.5 μM JQ1 (lane 2), 2.5 μM OTX015 (lane 3), 25 μM MS436 (lane 4) or 5 μM IBET151 (lane 5). Lanes 6–10 show extracts pretreated with RNase A to show activity due to RNase sensitive telomerase enzyme. ( B ) Quantification of telomerase processivity and ( C ) Quantification of telomerase catalytic activity as described in ‘Materials and Methods’ section. Values in (B) and (C) are averages of two technical replicates. Error bars in (C) represent the standard deviation.

Techniques Used: Inhibition, Activity Assay, Telomerase Assay, Standard Deviation

36) Product Images from "Double-Stranded RNA Derived from Lactic Acid Bacteria Augments Th1 Immunity via Interferon-β from Human Dendritic Cells"

Article Title: Double-Stranded RNA Derived from Lactic Acid Bacteria Augments Th1 Immunity via Interferon-β from Human Dendritic Cells

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.00027

Double-stranded RNA (dsRNA) in lactic acid bacteria (LAB) induce interleukin-12 (IL-12) secretion through IFN-β from peripheral blood mononuclear cells (PBMCs). (A) dsRNA in P. acidilactici strain K15 was quantified by sandwich ELISA. Heat-killed K15 was treated with RNase A in the absence of NaCl (0 M NaCl) to degrade ssRNA and dsRNA or under 0.3 M NaCl for the degradation of ssRNA alone. Data are the mean ± SD of triplicates and are representative of two independent experiments. (B) PBMCs were cultured in medium alone (−) or stimulated with untreated or RNase A-treated heat-killed LAB strains for 24 h. Tested strains are described in Table 1 . IL-12 concentration in culture medium was quantified by ELISA. Data are the mean ± SD of triplicates and are representative of two different donors (ND: not detected). ** p
Figure Legend Snippet: Double-stranded RNA (dsRNA) in lactic acid bacteria (LAB) induce interleukin-12 (IL-12) secretion through IFN-β from peripheral blood mononuclear cells (PBMCs). (A) dsRNA in P. acidilactici strain K15 was quantified by sandwich ELISA. Heat-killed K15 was treated with RNase A in the absence of NaCl (0 M NaCl) to degrade ssRNA and dsRNA or under 0.3 M NaCl for the degradation of ssRNA alone. Data are the mean ± SD of triplicates and are representative of two independent experiments. (B) PBMCs were cultured in medium alone (−) or stimulated with untreated or RNase A-treated heat-killed LAB strains for 24 h. Tested strains are described in Table 1 . IL-12 concentration in culture medium was quantified by ELISA. Data are the mean ± SD of triplicates and are representative of two different donors (ND: not detected). ** p

Techniques Used: Sandwich ELISA, Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay

Th1 cell differentiation is induced by dendritic cells (DCs) in response to double-stranded RNA (dsRNA) in lactic acid bacteria (LAB).  (A)  BDCA1 +  DCs (mDC1) were cultured in medium alone (−) or stimulated with untreated or RNase A-treated (0 M or 0.3 M NaCl) heat-killed K15 for 24 h. Interleukin-12 (IL-12) concentrations in culture medium were quantified by ELISA. Data are the mean ± SD of triplicates and are representative of two different donors. ** p
Figure Legend Snippet: Th1 cell differentiation is induced by dendritic cells (DCs) in response to double-stranded RNA (dsRNA) in lactic acid bacteria (LAB). (A) BDCA1 + DCs (mDC1) were cultured in medium alone (−) or stimulated with untreated or RNase A-treated (0 M or 0.3 M NaCl) heat-killed K15 for 24 h. Interleukin-12 (IL-12) concentrations in culture medium were quantified by ELISA. Data are the mean ± SD of triplicates and are representative of two different donors. ** p

Techniques Used: Cell Differentiation, Cell Culture, Enzyme-linked Immunosorbent Assay

37) Product Images from "Cellular RelB interacts with the transactivator Tat and enhance HIV-1 expression"

Article Title: Cellular RelB interacts with the transactivator Tat and enhance HIV-1 expression

Journal: Retrovirology

doi: 10.1186/s12977-018-0447-9

Interaction between HIV-1 Tat and RelB. a , b Myc-Tat (3 μg) was transfected into HEK 293T cells (4 × 10 6 ) together with empty vectors (control) (3 μg) or pFlag-RelB (3 μg) Co-immunoprecipitation was performed with anti-Flag ( a ) or anti-Myc ( b ) antibodies. Samples of both cell lysates and immunoprecipitates were subjected to western blotting and probed with rabbit anti-Myc and anti-Flag antibodies. c Co-IP of endogenous RelB and ectopically expressed Tat. The lysate from HA-Tat-expressing HeLa cells (4 × 10 6 ) was immunoprecipitated with mouse anti-HA antibodies, and the precipitated proteins were examined with western blotting. d Effect of RNases on the association of endogenous RelB and ectopically expressed Tat. Lysates (in the presence or absence of RNase A [5 μg/ml]) of HA-Tat expressing HeLa cells (4 × 10 6 ) were immunoprecipitated with control rabbit IgG or rabbit anti-RelB antibodies. Samples from cell lysates and immunoprecipitates were subjected to western blotting. e Tat partially co-localizes with RelB. HeLa cells (0.1 × 10 6 ) were transfected with HA-Tat (200 ng) and Flag-RelB (200 ng) plasmid DNA. Indirect IFA was performed to detect HA-Tat (with rabbit anti-HA antibody and TRITC-conjugated goat anti rabbit secondary antibody) and Flag-RelB (with mouse anti-Flag antibody and FITC-conjugated goat anti mouse secondary antibody). Nuclei were visualized with DAPI staining. Representative images are shown. The inset shows a higher magnification of the boxed area
Figure Legend Snippet: Interaction between HIV-1 Tat and RelB. a , b Myc-Tat (3 μg) was transfected into HEK 293T cells (4 × 10 6 ) together with empty vectors (control) (3 μg) or pFlag-RelB (3 μg) Co-immunoprecipitation was performed with anti-Flag ( a ) or anti-Myc ( b ) antibodies. Samples of both cell lysates and immunoprecipitates were subjected to western blotting and probed with rabbit anti-Myc and anti-Flag antibodies. c Co-IP of endogenous RelB and ectopically expressed Tat. The lysate from HA-Tat-expressing HeLa cells (4 × 10 6 ) was immunoprecipitated with mouse anti-HA antibodies, and the precipitated proteins were examined with western blotting. d Effect of RNases on the association of endogenous RelB and ectopically expressed Tat. Lysates (in the presence or absence of RNase A [5 μg/ml]) of HA-Tat expressing HeLa cells (4 × 10 6 ) were immunoprecipitated with control rabbit IgG or rabbit anti-RelB antibodies. Samples from cell lysates and immunoprecipitates were subjected to western blotting. e Tat partially co-localizes with RelB. HeLa cells (0.1 × 10 6 ) were transfected with HA-Tat (200 ng) and Flag-RelB (200 ng) plasmid DNA. Indirect IFA was performed to detect HA-Tat (with rabbit anti-HA antibody and TRITC-conjugated goat anti rabbit secondary antibody) and Flag-RelB (with mouse anti-Flag antibody and FITC-conjugated goat anti mouse secondary antibody). Nuclei were visualized with DAPI staining. Representative images are shown. The inset shows a higher magnification of the boxed area

Techniques Used: Transfection, Immunoprecipitation, Western Blot, Co-Immunoprecipitation Assay, Expressing, Plasmid Preparation, Immunofluorescence, Staining

38) Product Images from "Hadaka Virus 1: a Capsidless Eleven-Segmented Positive-Sense Single-Stranded RNA Virus from a Phytopathogenic Fungus, Fusarium oxysporum"

Article Title: Hadaka Virus 1: a Capsidless Eleven-Segmented Positive-Sense Single-Stranded RNA Virus from a Phytopathogenic Fungus, Fusarium oxysporum

Journal: mBio

doi: 10.1128/mBio.00450-20

RNase A treatment of mycelial extracts containing viral RNAs. Three F. oxysporum strains (a virus-free isolate [7n-cf1/VF], a HadV1-infected isolate [7n/HadV1], and a FoCV1-infected isolate [A-60/FoCV1]) and two P. janthinellum strains (a virus-free isolate [A-58-cf1/VF] and a PjPmV1-infected isolate [A-58/cf1]) were used. (A) Flow of the experiment. (B) Susceptibility of viral RNAs to RNase A. Electrophoretic profiles of viral dsRNA (top) and RT-PCR products from total RNA (bottom) detected before and after RNase A treatment are shown. Host mRNAs ( F. oxysporum ef1 α and P. janthinellum benA ) were targeted in parallel by RT-PCR.
Figure Legend Snippet: RNase A treatment of mycelial extracts containing viral RNAs. Three F. oxysporum strains (a virus-free isolate [7n-cf1/VF], a HadV1-infected isolate [7n/HadV1], and a FoCV1-infected isolate [A-60/FoCV1]) and two P. janthinellum strains (a virus-free isolate [A-58-cf1/VF] and a PjPmV1-infected isolate [A-58/cf1]) were used. (A) Flow of the experiment. (B) Susceptibility of viral RNAs to RNase A. Electrophoretic profiles of viral dsRNA (top) and RT-PCR products from total RNA (bottom) detected before and after RNase A treatment are shown. Host mRNAs ( F. oxysporum ef1 α and P. janthinellum benA ) were targeted in parallel by RT-PCR.

Techniques Used: Infection, Reverse Transcription Polymerase Chain Reaction

39) 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:

40) 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

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In Vitro:

Article Title: The 125th Lys and 145th Thr Amino Acids in the GTPase Domain of Goose Mx Confer Its Antiviral Activity against the Tembusu Virus
Article Snippet: .. Immunological Characteristic Research In Vitro The polyinosinic polycytidylic acid (Poly(I:C); Sigma, St. Louis, MO, USA) can mimic the treatment of double-stranded RNA. ..

Cell Culture:

Article Title: Long Interleukin-22 Binding Protein Isoform-1 Is an Intracellular Activator of the Unfolded Protein Response
Article Snippet: .. Maturation of moDCs was induced by adding IFN-γ (Peprotech, AF-300-02; 500 pg/ ml) for overnight priming (16 h) followed by late addition of LPS for 6 hrs (Sigma, L6143; 2 μg/ml) to the culture or, alternatively by addition of CpG (Miltenyi, 130-100-243; 1 μg/ml) or LPS/Poly(I:C) (Sigma, P1530; 1 μg/ml) for 6 h. HEK293, HeLa and A549 were cultured in DMEM supplemented with 10% FBS and 2 mM L-glutamine; U937 cells were cultured in RPMI supplemented with 0.05 mM β-mercaptoethanol, 10 % FBS and 2 mM L-glutamine. .. FACS of Monocytes and Flow Cytometry of moDCs PBMCs were obtained from freshly isolated buffy coats of healthy donors using Ficoll-Hypaque gradient (ThermoFisher).

Mouse Assay:

Article Title: YK-4-279 effectively antagonizes EWS-FLI1 induced leukemia in a transgenic mouse model
Article Snippet: .. E/F; Mx1-cre mice were injected with 1 mg pIpC (Sigma, Cat No. P0913) at 1 month of age to induce Mx1 promoter. .. Blood was drawn from submandibular vein using 3 mm sterile animal lancets (MEDIpoint, Cat No. Goldenrod 3 mm) in microtainer tubes with EDTA (BD, Cat No. 365973) before pIpC injection and weekly afterwards.

Concentration Assay:

Article Title: IL-8 secretion in primary cultures of prostate cells is associated with prostate cancer aggressiveness
Article Snippet: .. After 24 hours, 50 μL of KSFM containing 20 μg/mL of polyinosinic:polycytidylic acid (poly(I:C); Sigma-Aldrich) was added in half of the wells to reach a final concentration of 10 μg/mL, while 50 μL of KSFM alone was added to control wells. .. After 20 hours, cell supernatants were collected for IL-8 measurement and total DNA was measured according to the DRAQ5® LI-COR® protocol (LI-COR Biosciences, Lincoln, NE, USA), to account for the number of cells present per well.

Article Title: Human Nup98 regulates the localization and activity of DExH/D-box helicase DHX9
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Incubation:

Article Title: Human Nup98 regulates the localization and activity of DExH/D-box helicase DHX9
Article Snippet: .. After washing to remove unbound protein, beads were resuspended in a total volume of 1.2 ml of PBS-T and 400 µl aliquots were incubated for 10 min at room temperature with either RNase A (final concentration 100 µg/ml), poly I:C RNA (Sigma-Aldrich P1530) (final concentration 100 µg/ml), or buffer alone. .. In parallel, GST-tagged Nup98 (1.2 nmoles in 200 µl of PBS-T) and purified GST (1.2 nmoles in 200 µl of PBS-T) were similarly treated with RNase A, poly I:C RNA, or buffer alone.

Injection:

Article Title: YK-4-279 effectively antagonizes EWS-FLI1 induced leukemia in a transgenic mouse model
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  • 99
    Millipore ribonuclease p1
    Methylated nucleoside content of total RNA isolated from sporulating and non-sporulating yeast. ( A ) Yeast grown in methionine-deficient medium were labeled with [ 3 H- methyl -]methionine. Total RNA was prepared, digested with <t>ribonuclease</t> P1 and nucleoside pyrophosphatase, and dephosphorylated with alkaline phosphatase. The sample was then chromatographed as described in the text. The positions of the predominant 2′- O -methylribonucleosides (N m ) are shown, as is the position of the much smaller m 6 A peak. The data shown represent the mean of nine separate experiments (control) and 10 experiments (sporulated). Each sample consisted of ∼10 µg of total RNA, containing ∼100 000 c.p.m. of incorporated 3 H. All data were standardized to account for differences in the total counts loaded onto the column. ( B ) The same data are replotted on an expanded scale to better illustrate the m 6 A peak. ( C ) The radioactivity in the m 6 A peak is represented graphically. Error bars represent the standard deviation from the mean for each group of samples.
    Ribonuclease P1, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore rnase a
    Polytene chromosome spreads of  D. melanogaster  wild type were treated with RNase A/RNase H mixture followed by proteinase K digestion in a time course experiment and subsequent immunological detection of triple-stranded DNA. DAPI staining (blue signal) and antibody labelling (red signal) were superimposed. Scale bar represents 25 µm.
    Rnase A, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 327 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore rnase t1
    Determination of minimum RNA fragment bound by MSY2 and MSY4. (A) Schematic depiction of Prm1 1–37wt RNA and four mutant RNAs engineered such that <t>RNase</t> T1 treatment produces different-size RNA fragments containing the YRS. Arrows, RNase T1 cleavage sites. The single nucleotide substitution in T1.8m is underlined. (B) Urea gel analysis of RNase T1 precut RNAs. Top arrow, size of the RNAs prior to cutting (43 nt). Upon treatment with RNase T1, YRS-containing RNA fragments of 12, 10, and 8 nt are released. No uncut RNA of 43 nt is seen in the cut-RNA lanes. (C) UV cross-linking analysis of MSY2 and MSY4 binding of the RNAs depicted in panels A and B. MSY2 and MSY4 were able to bind the T1.12, T1.10, and T1.8 RNA substrates before and after treatment with RNase T1.
    Rnase T1, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 132 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Methylated nucleoside content of total RNA isolated from sporulating and non-sporulating yeast. ( A ) Yeast grown in methionine-deficient medium were labeled with [ 3 H- methyl -]methionine. Total RNA was prepared, digested with ribonuclease P1 and nucleoside pyrophosphatase, and dephosphorylated with alkaline phosphatase. The sample was then chromatographed as described in the text. The positions of the predominant 2′- O -methylribonucleosides (N m ) are shown, as is the position of the much smaller m 6 A peak. The data shown represent the mean of nine separate experiments (control) and 10 experiments (sporulated). Each sample consisted of ∼10 µg of total RNA, containing ∼100 000 c.p.m. of incorporated 3 H. All data were standardized to account for differences in the total counts loaded onto the column. ( B ) The same data are replotted on an expanded scale to better illustrate the m 6 A peak. ( C ) The radioactivity in the m 6 A peak is represented graphically. Error bars represent the standard deviation from the mean for each group of samples.

    Journal: Nucleic Acids Research

    Article Title: Induction of sporulation in Saccharomyces cerevisiae leads to the formation of N6-methyladenosine in mRNA: a potential mechanism for the activity of the IME4 gene

    doi:

    Figure Lengend Snippet: Methylated nucleoside content of total RNA isolated from sporulating and non-sporulating yeast. ( A ) Yeast grown in methionine-deficient medium were labeled with [ 3 H- methyl -]methionine. Total RNA was prepared, digested with ribonuclease P1 and nucleoside pyrophosphatase, and dephosphorylated with alkaline phosphatase. The sample was then chromatographed as described in the text. The positions of the predominant 2′- O -methylribonucleosides (N m ) are shown, as is the position of the much smaller m 6 A peak. The data shown represent the mean of nine separate experiments (control) and 10 experiments (sporulated). Each sample consisted of ∼10 µg of total RNA, containing ∼100 000 c.p.m. of incorporated 3 H. All data were standardized to account for differences in the total counts loaded onto the column. ( B ) The same data are replotted on an expanded scale to better illustrate the m 6 A peak. ( C ) The radioactivity in the m 6 A peak is represented graphically. Error bars represent the standard deviation from the mean for each group of samples.

    Article Snippet: Labeled yeast RNA was digested with 10 µg of ribonuclease P1 (Calbiochem) and 0.125 U nucleotide pyrophosphatase (Sigma) in 5 mm sodium acetate pH 6.0, 1 mM MgCl2 , in a final volume of 50 µl for 4 h at 37°C.

    Techniques: Methylation, Isolation, Labeling, Radioactivity, Standard Deviation

    Polytene chromosome spreads of  D. melanogaster  wild type were treated with RNase A/RNase H mixture followed by proteinase K digestion in a time course experiment and subsequent immunological detection of triple-stranded DNA. DAPI staining (blue signal) and antibody labelling (red signal) were superimposed. Scale bar represents 25 µm.

    Journal: Cells

    Article Title: Triple-Helical DNA in Drosophila Heterochromatin

    doi: 10.3390/cells7120227

    Figure Lengend Snippet: Polytene chromosome spreads of D. melanogaster wild type were treated with RNase A/RNase H mixture followed by proteinase K digestion in a time course experiment and subsequent immunological detection of triple-stranded DNA. DAPI staining (blue signal) and antibody labelling (red signal) were superimposed. Scale bar represents 25 µm.

    Article Snippet: For RNase treatment, chromosome spreads were rehydrated in 1× TBS followed by incubation at room temperature with RNase A (Calbiochem, San Diego, CA, USA) diluted (0.2 mg/mL) in 2× SSC for 2 h. Additional enzymatic treatments were carried out at room temperature with a mixture of RNase A (Calbiochem, San Diego, CA, USA, 0.2 mg/mL) and RNase H (GE Healthcare, Chicago, IL, USA, 1 unit per slide) diluted in 1× PBS.

    Techniques: Staining

    Determination of minimum RNA fragment bound by MSY2 and MSY4. (A) Schematic depiction of Prm1 1–37wt RNA and four mutant RNAs engineered such that RNase T1 treatment produces different-size RNA fragments containing the YRS. Arrows, RNase T1 cleavage sites. The single nucleotide substitution in T1.8m is underlined. (B) Urea gel analysis of RNase T1 precut RNAs. Top arrow, size of the RNAs prior to cutting (43 nt). Upon treatment with RNase T1, YRS-containing RNA fragments of 12, 10, and 8 nt are released. No uncut RNA of 43 nt is seen in the cut-RNA lanes. (C) UV cross-linking analysis of MSY2 and MSY4 binding of the RNAs depicted in panels A and B. MSY2 and MSY4 were able to bind the T1.12, T1.10, and T1.8 RNA substrates before and after treatment with RNase T1.

    Journal: Molecular and Cellular Biology

    Article Title: MSY2 and MSY4 Bind a Conserved Sequence in the 3? Untranslated Region of Protamine 1 mRNA In Vitro and In Vivo

    doi: 10.1128/MCB.21.20.7010-7019.2001

    Figure Lengend Snippet: Determination of minimum RNA fragment bound by MSY2 and MSY4. (A) Schematic depiction of Prm1 1–37wt RNA and four mutant RNAs engineered such that RNase T1 treatment produces different-size RNA fragments containing the YRS. Arrows, RNase T1 cleavage sites. The single nucleotide substitution in T1.8m is underlined. (B) Urea gel analysis of RNase T1 precut RNAs. Top arrow, size of the RNAs prior to cutting (43 nt). Upon treatment with RNase T1, YRS-containing RNA fragments of 12, 10, and 8 nt are released. No uncut RNA of 43 nt is seen in the cut-RNA lanes. (C) UV cross-linking analysis of MSY2 and MSY4 binding of the RNAs depicted in panels A and B. MSY2 and MSY4 were able to bind the T1.12, T1.10, and T1.8 RNA substrates before and after treatment with RNase T1.

    Article Snippet: Samples were then sequentially treated with 2 μl of RNase T1 (Calbiochem) at 2 U/μl and 4 μl of heparin (Sigma) at 5 mg/ml, each for 10 min at room temperature.

    Techniques: Mutagenesis, Binding Assay

    Mutational analysis of the HSUR 1 ARE. ( A ) Sequences of the six HSUR 1 point mutants, M1–M6, with the sites of U → G mutation underlined. ( B ) RNA levels of the HSUR 1 mutants. Wild-type HSUR 1 and mutants M1–M6, all driven by the same U1 promoter, were each transiently cotransfected with HSUR 3 into mouse L929 cells, and the RNA levels were assayed by T1 RNase protection (lanes 1–7, respectively). The antisense HSUR 1 probe covered a 120-nucleotide region at the 3′ end, which is common to wild-type HSUR 1 and all six mutants. When quantitated and normalized to HSUR 3, the mutant M1–M6 levels were found to be 7.0-, 10.1-, 1.8-, 5.8-, 6.4-, and 6.1-fold that of wild-type HSUR 1.

    Journal: Genes & Development

    Article Title: AU-rich elements target small nuclear RNAs as well as mRNAs for rapid degradation

    doi:

    Figure Lengend Snippet: Mutational analysis of the HSUR 1 ARE. ( A ) Sequences of the six HSUR 1 point mutants, M1–M6, with the sites of U → G mutation underlined. ( B ) RNA levels of the HSUR 1 mutants. Wild-type HSUR 1 and mutants M1–M6, all driven by the same U1 promoter, were each transiently cotransfected with HSUR 3 into mouse L929 cells, and the RNA levels were assayed by T1 RNase protection (lanes 1–7, respectively). The antisense HSUR 1 probe covered a 120-nucleotide region at the 3′ end, which is common to wild-type HSUR 1 and all six mutants. When quantitated and normalized to HSUR 3, the mutant M1–M6 levels were found to be 7.0-, 10.1-, 1.8-, 5.8-, 6.4-, and 6.1-fold that of wild-type HSUR 1.

    Article Snippet: T1 RNase protection assays were performed as described (S. ), with the following modifications: DNase-treated RNA (5–10 μg for snRNA, 20–30 μg for mRNA) was combined with 2 × 105 to 4 × 105 cpm of the appropriate [α-32 P]UTP-labeled antisense probe, heated at 85°C for 5 min, incubated at 45°C overnight to allow annealing, and then digested with T1 RNase (1 U/10 μg of RNA; Calbiochem) at 30°C for 1 hr.

    Techniques: Mutagenesis

    AUUUA repeats target other snRNAs for rapid decay. ( A ) The 5′ ARE of wild-type HSUR 2 and its mutants. The single guanosine that interrupts the AUUUA repeat sequence in HSUR 2 was mutated to UA, CC, and CA (underlined) in HSUR 2 M1, HSUR 2 M2, and HSUR 2 M3, respectively. ( B ) RNA levels of wild-type and mutant HSUR 2s. Wild-type HSUR 2 or mutant M1, M2, or M3, all controlled by the same U1 promoter, were each transiently cotransfected with HSUR 3 into mouse L929 cells and analyzed by T1 RNase protection. The level of mutant M1 (lane 2 ), which has four tandem copies of AUUU, is one-fifth that of wild-type HSUR 2 (lane 1 ), whereas controls M2 and M3 (lanes 3 and 4, respectively), which have been mutated at the same two positions as M1, have levels 1.3- and 1.1-fold of the wild-type HSUR 2, respectively. ( C ) The 5′-end sequences of wild-type U1 and two U1 mutants. The 5′ splice site recognition sequence of U1 and the sequences replacing it in the mutants are underlined. The AU3–U1 mutant has four tandem copies of AUUU, whereas in the AGU–U1 mutant, there are four AUUU repeats interrupted by 3 Gs (the same sequence as in HSUR 1 M2). ( D ) Levels of AU3–U1 and AGU–U1 in duplicate transfection experiments. Each U1 mutant was transiently cotransfected with HSUR 3 into mouse L929 cells. U1 RNA levels were analyzed by primer extension using an oligonucleotide complementary to the most 3′ 20 nucleotides of U1 (nucleotides 155–164), whereas HSUR 3 was assayed by T1 RNase protection assay as above. When normalized to HSUR 3 and averaged between the duplicate transfection experiments, the AU3–U1 level was found to be one-fourth that of AGU–U1.

    Journal: Genes & Development

    Article Title: AU-rich elements target small nuclear RNAs as well as mRNAs for rapid degradation

    doi:

    Figure Lengend Snippet: AUUUA repeats target other snRNAs for rapid decay. ( A ) The 5′ ARE of wild-type HSUR 2 and its mutants. The single guanosine that interrupts the AUUUA repeat sequence in HSUR 2 was mutated to UA, CC, and CA (underlined) in HSUR 2 M1, HSUR 2 M2, and HSUR 2 M3, respectively. ( B ) RNA levels of wild-type and mutant HSUR 2s. Wild-type HSUR 2 or mutant M1, M2, or M3, all controlled by the same U1 promoter, were each transiently cotransfected with HSUR 3 into mouse L929 cells and analyzed by T1 RNase protection. The level of mutant M1 (lane 2 ), which has four tandem copies of AUUU, is one-fifth that of wild-type HSUR 2 (lane 1 ), whereas controls M2 and M3 (lanes 3 and 4, respectively), which have been mutated at the same two positions as M1, have levels 1.3- and 1.1-fold of the wild-type HSUR 2, respectively. ( C ) The 5′-end sequences of wild-type U1 and two U1 mutants. The 5′ splice site recognition sequence of U1 and the sequences replacing it in the mutants are underlined. The AU3–U1 mutant has four tandem copies of AUUU, whereas in the AGU–U1 mutant, there are four AUUU repeats interrupted by 3 Gs (the same sequence as in HSUR 1 M2). ( D ) Levels of AU3–U1 and AGU–U1 in duplicate transfection experiments. Each U1 mutant was transiently cotransfected with HSUR 3 into mouse L929 cells. U1 RNA levels were analyzed by primer extension using an oligonucleotide complementary to the most 3′ 20 nucleotides of U1 (nucleotides 155–164), whereas HSUR 3 was assayed by T1 RNase protection assay as above. When normalized to HSUR 3 and averaged between the duplicate transfection experiments, the AU3–U1 level was found to be one-fourth that of AGU–U1.

    Article Snippet: T1 RNase protection assays were performed as described (S. ), with the following modifications: DNase-treated RNA (5–10 μg for snRNA, 20–30 μg for mRNA) was combined with 2 × 105 to 4 × 105 cpm of the appropriate [α-32 P]UTP-labeled antisense probe, heated at 85°C for 5 min, incubated at 45°C overnight to allow annealing, and then digested with T1 RNase (1 U/10 μg of RNA; Calbiochem) at 30°C for 1 hr.

    Techniques: Sequencing, Mutagenesis, Transfection, Rnase Protection Assay

    ARE-mediated HSUR 1 degradation in vivo. ( A ). ( B ) T1 RNase protection analysis of wild-type and mutant HSUR 1 levels in transient transfection assays. The pUC–U1–HSUR 1 constructs were transiently cotransfected with a pUC–U1–HSUR 3 plasmid into mouse L929 cells (see Materials and Methods). Total RNA collected 48 hr after transfection was subjected to RNase T1 protection assays with wild-type and mutant HSUR 1 antisense RNA (lanes 2 and 3, respectively), together with antisense HSUR 3 RNA as an internal control. One-fiftieth of the amount of the anti-wild-type and anti-mutant HSUR 1 RNA probes used is shown in lanes 4 and 5. The data were quantitated with a Molecular Dynamics PhosphorImager and normalized to HSUR 3. Wild-type HSUR 1 levels were reproducibly one-eighth of those of mutant HSUR 1. ( C ) Whole-cell run-on assays of wild-type and mutant HSUR 1 transcription. The pUC–U1–HSUR 1 construct containing wild-type or mutant HSUR 1 sequences was cotransfected with pUC–U1–HSUR 3 into L cells, and whole-cell run-on assays were performed (see Materials and Methods). Total RNA was hybridized to nylon membranes that had been dot-blotted with wild-type ( top ) or mutant ( bottom ) HSUR 1 and HSUR 3 DNA fragments. Untransfected HSUR 4 DNA was also dotted as a negative control. The patterns of dots are illustrated at right; hybridizations with the run-on RNAs are at left. After quantitation and normalization against the cotransfected positive control HSUR 3 (also subtracting the untransfected negative control HSUR 4), the wild-type HSUR 1 ( left dot, top ) and the mutant ( left dot, bottom ) were found to have similar transcription rates (wild type:mutant = 0.95). ( D ) Immunoprecipitation of wild-type and mutant HSUR 1 from transfected mouse L929 cells. L cells were transiently transfected with the pUC–U1 constructs containing wild-type or mutant HSUR 1 genes, and whole-cell extracts were prepared by sonication. Equal amounts of extract were precipitated with anti-Sm monoclonal antibody Y12 or anti-U1 70K monoclonal antibody H111 as a control. RNA was harvested from the immunoprecipitation pellets (lanes 1,3,5,7 ) and supernatants (lanes 2,4,6,8 ), and wild-type ( left ) and mutant ( right ) HSUR 1 were assayed by T1 RNase protection. For both wild-type and mutant HSUR 1s, > 90% of the RNA was in the anti-Sm precipitate (lanes 3,7 ), whereas > 99% of the HSUR 1s remained in the supernatant with the anti-U1 70K antibody (lanes 2,6 ).

    Journal: Genes & Development

    Article Title: AU-rich elements target small nuclear RNAs as well as mRNAs for rapid degradation

    doi:

    Figure Lengend Snippet: ARE-mediated HSUR 1 degradation in vivo. ( A ). ( B ) T1 RNase protection analysis of wild-type and mutant HSUR 1 levels in transient transfection assays. The pUC–U1–HSUR 1 constructs were transiently cotransfected with a pUC–U1–HSUR 3 plasmid into mouse L929 cells (see Materials and Methods). Total RNA collected 48 hr after transfection was subjected to RNase T1 protection assays with wild-type and mutant HSUR 1 antisense RNA (lanes 2 and 3, respectively), together with antisense HSUR 3 RNA as an internal control. One-fiftieth of the amount of the anti-wild-type and anti-mutant HSUR 1 RNA probes used is shown in lanes 4 and 5. The data were quantitated with a Molecular Dynamics PhosphorImager and normalized to HSUR 3. Wild-type HSUR 1 levels were reproducibly one-eighth of those of mutant HSUR 1. ( C ) Whole-cell run-on assays of wild-type and mutant HSUR 1 transcription. The pUC–U1–HSUR 1 construct containing wild-type or mutant HSUR 1 sequences was cotransfected with pUC–U1–HSUR 3 into L cells, and whole-cell run-on assays were performed (see Materials and Methods). Total RNA was hybridized to nylon membranes that had been dot-blotted with wild-type ( top ) or mutant ( bottom ) HSUR 1 and HSUR 3 DNA fragments. Untransfected HSUR 4 DNA was also dotted as a negative control. The patterns of dots are illustrated at right; hybridizations with the run-on RNAs are at left. After quantitation and normalization against the cotransfected positive control HSUR 3 (also subtracting the untransfected negative control HSUR 4), the wild-type HSUR 1 ( left dot, top ) and the mutant ( left dot, bottom ) were found to have similar transcription rates (wild type:mutant = 0.95). ( D ) Immunoprecipitation of wild-type and mutant HSUR 1 from transfected mouse L929 cells. L cells were transiently transfected with the pUC–U1 constructs containing wild-type or mutant HSUR 1 genes, and whole-cell extracts were prepared by sonication. Equal amounts of extract were precipitated with anti-Sm monoclonal antibody Y12 or anti-U1 70K monoclonal antibody H111 as a control. RNA was harvested from the immunoprecipitation pellets (lanes 1,3,5,7 ) and supernatants (lanes 2,4,6,8 ), and wild-type ( left ) and mutant ( right ) HSUR 1 were assayed by T1 RNase protection. For both wild-type and mutant HSUR 1s, > 90% of the RNA was in the anti-Sm precipitate (lanes 3,7 ), whereas > 99% of the HSUR 1s remained in the supernatant with the anti-U1 70K antibody (lanes 2,6 ).

    Article Snippet: T1 RNase protection assays were performed as described (S. ), with the following modifications: DNase-treated RNA (5–10 μg for snRNA, 20–30 μg for mRNA) was combined with 2 × 105 to 4 × 105 cpm of the appropriate [α-32 P]UTP-labeled antisense probe, heated at 85°C for 5 min, incubated at 45°C overnight to allow annealing, and then digested with T1 RNase (1 U/10 μg of RNA; Calbiochem) at 30°C for 1 hr.

    Techniques: In Vivo, Mutagenesis, Transfection, Construct, Plasmid Preparation, Negative Control, Quantitation Assay, Positive Control, Immunoprecipitation, Sonication