Structured Review

PerkinElmer α 32 p gtp
YgdH binds (p)ppGpp antagonistically with magnesium. (A) Competition assay of purified YgdH protein (20 μM) binding a 1:1 mixture of ppGpp and pppGpp (2 nM [each]) in the absence or presence of EDTA. Representative DRaCALA spots and quantifications (average values for bound fractions and standards errors of the means [SEM]) of binding signals are shown. (B) Thin-layer chromatography (TLC) of DRaCALA binding reactions determined by using 1.5 M K 2 HPO 4 (pH 3.4) as the mobile phase. Binding reactions performed with purified MutT, Der, or YgdH were run in parallel with standards of [α- 32 <t>P]GTP</t> and a mixture of [α- 32 P]ppGpp and [α- 32 P]pppGpp (2 nM [each]). (C) Binding curves and K d determinations for YgdH interacting with α- 32 P-labeled ppGpp, pppGpp, and GTP (2 nM [each]) without or with 1.5 mM MgCl 2 . The apparent K d values corresponding to each protein-ligand interaction are shown. (D) Magnesium (0 to 10.15 mM) IC 50 determinations of binding of [α- 32 P]ppGpp (2 nM) to YgdH (50 μM). IC 50 values are shown. (E) Competition assay of YgdH (50 μM) binding [α- 32 P]ppGpp (2 nM) in the presence of 100 μM cold competitors [including (p)ppGpp and the substrates of YgdH (GMP, AMP, and IMP)] without or with 1.5 mM magnesium.
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1) Product Images from "Novel (p)ppGpp Binding and Metabolizing Proteins of Escherichia coli"

Article Title: Novel (p)ppGpp Binding and Metabolizing Proteins of Escherichia coli

Journal: mBio

doi: 10.1128/mBio.02188-17

YgdH binds (p)ppGpp antagonistically with magnesium. (A) Competition assay of purified YgdH protein (20 μM) binding a 1:1 mixture of ppGpp and pppGpp (2 nM [each]) in the absence or presence of EDTA. Representative DRaCALA spots and quantifications (average values for bound fractions and standards errors of the means [SEM]) of binding signals are shown. (B) Thin-layer chromatography (TLC) of DRaCALA binding reactions determined by using 1.5 M K 2 HPO 4 (pH 3.4) as the mobile phase. Binding reactions performed with purified MutT, Der, or YgdH were run in parallel with standards of [α- 32 P]GTP and a mixture of [α- 32 P]ppGpp and [α- 32 P]pppGpp (2 nM [each]). (C) Binding curves and K d determinations for YgdH interacting with α- 32 P-labeled ppGpp, pppGpp, and GTP (2 nM [each]) without or with 1.5 mM MgCl 2 . The apparent K d values corresponding to each protein-ligand interaction are shown. (D) Magnesium (0 to 10.15 mM) IC 50 determinations of binding of [α- 32 P]ppGpp (2 nM) to YgdH (50 μM). IC 50 values are shown. (E) Competition assay of YgdH (50 μM) binding [α- 32 P]ppGpp (2 nM) in the presence of 100 μM cold competitors [including (p)ppGpp and the substrates of YgdH (GMP, AMP, and IMP)] without or with 1.5 mM magnesium.
Figure Legend Snippet: YgdH binds (p)ppGpp antagonistically with magnesium. (A) Competition assay of purified YgdH protein (20 μM) binding a 1:1 mixture of ppGpp and pppGpp (2 nM [each]) in the absence or presence of EDTA. Representative DRaCALA spots and quantifications (average values for bound fractions and standards errors of the means [SEM]) of binding signals are shown. (B) Thin-layer chromatography (TLC) of DRaCALA binding reactions determined by using 1.5 M K 2 HPO 4 (pH 3.4) as the mobile phase. Binding reactions performed with purified MutT, Der, or YgdH were run in parallel with standards of [α- 32 P]GTP and a mixture of [α- 32 P]ppGpp and [α- 32 P]pppGpp (2 nM [each]). (C) Binding curves and K d determinations for YgdH interacting with α- 32 P-labeled ppGpp, pppGpp, and GTP (2 nM [each]) without or with 1.5 mM MgCl 2 . The apparent K d values corresponding to each protein-ligand interaction are shown. (D) Magnesium (0 to 10.15 mM) IC 50 determinations of binding of [α- 32 P]ppGpp (2 nM) to YgdH (50 μM). IC 50 values are shown. (E) Competition assay of YgdH (50 μM) binding [α- 32 P]ppGpp (2 nM) in the presence of 100 μM cold competitors [including (p)ppGpp and the substrates of YgdH (GMP, AMP, and IMP)] without or with 1.5 mM magnesium.

Techniques Used: Competitive Binding Assay, Purification, Binding Assay, Thin Layer Chromatography, Labeling

Translational GTPases are conserved targets of (p)ppGpp. (A) Competition assay of RF3 and Der (20 μM) and LepA (10 μM) binding [α- 32 P]ppGpp (2 nM) in the presence of cold competitors (100 μM). (B and C) Binding curves and K d determination of RF3 (B) and Der (C) binding of α- 32 P-labeled ppGpp, pppGpp, GTP, or GDP (2 nM [each]). At least three replicates were performed. The apparent K d values corresponding to each protein-ligand interaction are shown. (D) Dissociation curves for Der (50 μM) and [α- 32 P]ppGpp (2 nM) in the presence of either ppGpp or GTP (100 μM) (cold). (E and F) Binding curves and K d determination for DerG1 (E) and SaDer (F) binding α- 32 P-labeled ppGpp, pppGpp, or GTP (2 nM [each]). At least three replicates were performed. The apparent K d values are shown for each protein-ligand interaction.
Figure Legend Snippet: Translational GTPases are conserved targets of (p)ppGpp. (A) Competition assay of RF3 and Der (20 μM) and LepA (10 μM) binding [α- 32 P]ppGpp (2 nM) in the presence of cold competitors (100 μM). (B and C) Binding curves and K d determination of RF3 (B) and Der (C) binding of α- 32 P-labeled ppGpp, pppGpp, GTP, or GDP (2 nM [each]). At least three replicates were performed. The apparent K d values corresponding to each protein-ligand interaction are shown. (D) Dissociation curves for Der (50 μM) and [α- 32 P]ppGpp (2 nM) in the presence of either ppGpp or GTP (100 μM) (cold). (E and F) Binding curves and K d determination for DerG1 (E) and SaDer (F) binding α- 32 P-labeled ppGpp, pppGpp, or GTP (2 nM [each]). At least three replicates were performed. The apparent K d values are shown for each protein-ligand interaction.

Techniques Used: Competitive Binding Assay, Binding Assay, Labeling

HypB specifically binds (p)ppGpp with physiological affinity. (A) Competition assay of HypB (20 μM) binding α- 32 P-labeled ppGpp and pppGpp (2 nM [each]) in the presence of cold competitors (100 μM). (B) Binding curves and K d determination for HypB binding α- 32 P-labeled ppGpp, pppGpp, and GTP (2 nM [each]). Three replicates were performed, and the apparent K d values are indicated.
Figure Legend Snippet: HypB specifically binds (p)ppGpp with physiological affinity. (A) Competition assay of HypB (20 μM) binding α- 32 P-labeled ppGpp and pppGpp (2 nM [each]) in the presence of cold competitors (100 μM). (B) Binding curves and K d determination for HypB binding α- 32 P-labeled ppGpp, pppGpp, and GTP (2 nM [each]). Three replicates were performed, and the apparent K d values are indicated.

Techniques Used: Competitive Binding Assay, Binding Assay, Labeling

In vitro cleavage of ppGpp by MutT, NudG, NadR, and TrmE. (A) Competition assay of whole-cell lysates containing overexpressed MutT and NudG binding [α- 32 P]ppGpp (2 nM) in the presence of cold competitors (100 μM). (B) Competition assay of purified NadR (left) and TrmE (right) (20 μM [each]) binding α- 32 P-labeled ppGpp and pppGpp (2 nM [each]) in the presence of cold competitors (100 μM). (C) DRaCALA spots of purified proteins (10 μM) binding a mixture of α- 32 P-labeled ppGpp and pppGpp (2 nM [each]) in the absence or presence of EDTA (25 mM). (D) TLC assessment of cleavage products from the binding reactions described for panel C. A mixture of ppGpp and pppGpp was run as the standard, and both molecules are indicated. (E) Quantification of (p)ppGpp percentage determined as described for panel D. (F) Competition assay of whole-cell lysates containing overproduced MutT and NudG binding [α- 32 P](p)ppGpp (2 nM) in the presence of cold competitors and their native substrates (100 μM [each]). Representative DRaCALA spots are shown. 8OdG, 8-oxo-dGTP; 8OG, 8-oxo-GTP; 2OdA, 2-hydroxyl-dATP; 2OA, 2-hydroxyl-ATP. (G) TLC assessment of cleavage products of [α- 32 P]ppGpp (10 nM) determined using purified MutT and NudG (1 μM) in the presence of cold competitors (100 μM) or excess EDTA (25 mM). Samples were incubated at 30°C for 10 min (or 1 h; see Fig. S6D ), and reactions were stopped by addition of excess EDTA (25 mM). pGp and ppGpp are indicated.
Figure Legend Snippet: In vitro cleavage of ppGpp by MutT, NudG, NadR, and TrmE. (A) Competition assay of whole-cell lysates containing overexpressed MutT and NudG binding [α- 32 P]ppGpp (2 nM) in the presence of cold competitors (100 μM). (B) Competition assay of purified NadR (left) and TrmE (right) (20 μM [each]) binding α- 32 P-labeled ppGpp and pppGpp (2 nM [each]) in the presence of cold competitors (100 μM). (C) DRaCALA spots of purified proteins (10 μM) binding a mixture of α- 32 P-labeled ppGpp and pppGpp (2 nM [each]) in the absence or presence of EDTA (25 mM). (D) TLC assessment of cleavage products from the binding reactions described for panel C. A mixture of ppGpp and pppGpp was run as the standard, and both molecules are indicated. (E) Quantification of (p)ppGpp percentage determined as described for panel D. (F) Competition assay of whole-cell lysates containing overproduced MutT and NudG binding [α- 32 P](p)ppGpp (2 nM) in the presence of cold competitors and their native substrates (100 μM [each]). Representative DRaCALA spots are shown. 8OdG, 8-oxo-dGTP; 8OG, 8-oxo-GTP; 2OdA, 2-hydroxyl-dATP; 2OA, 2-hydroxyl-ATP. (G) TLC assessment of cleavage products of [α- 32 P]ppGpp (10 nM) determined using purified MutT and NudG (1 μM) in the presence of cold competitors (100 μM) or excess EDTA (25 mM). Samples were incubated at 30°C for 10 min (or 1 h; see Fig. S6D ), and reactions were stopped by addition of excess EDTA (25 mM). pGp and ppGpp are indicated.

Techniques Used: In Vitro, Competitive Binding Assay, Binding Assay, Purification, Labeling, Thin Layer Chromatography, Incubation

GTP biosynthesis and salvage pathways are targeted by (p)ppGpp. (A) Schematic of purine biosynthesis pathways with (p)ppGpp targets highlighted by colored boxes. Green indicates E. coli targets identified here; blue indicates specific Bacillus / Staphylococcus targets; red indicates E. coli targets reported previously but not confirmed in this study; gray indicates a target found in E. coli , Bacillus , and Staphylococcus . G, guanine; X, xanthine; H, hypoxanthine; A, adenine; PRPP, phosphoribosyl pyrophosphate; Gln, glutamine. (B) Binding curves and apparent K d values for E. coli Gpt, Hpt, and Apt binding pppGpp and ppGpp (2 nM [each]). The average values for bound fractions and standard errors of the means (SEM) determined for at least three replicates were plotted and the curve-fitted and K d values determined as previously described ( 37 ). The apparent K d values corresponding to each protein-ligand interaction are shown. (C) Competition assay of Gpt, Hpt, and Apt (20 μM [each]) binding [α- 32 P]ppGpp (2 nM) in the presence of cold competitors (100 μM). The average values for bound fractions and standard errors of the means (SEM) determined for at least three replicates were plotted. Representative DRaCALA spots are shown above the respective diagrams.
Figure Legend Snippet: GTP biosynthesis and salvage pathways are targeted by (p)ppGpp. (A) Schematic of purine biosynthesis pathways with (p)ppGpp targets highlighted by colored boxes. Green indicates E. coli targets identified here; blue indicates specific Bacillus / Staphylococcus targets; red indicates E. coli targets reported previously but not confirmed in this study; gray indicates a target found in E. coli , Bacillus , and Staphylococcus . G, guanine; X, xanthine; H, hypoxanthine; A, adenine; PRPP, phosphoribosyl pyrophosphate; Gln, glutamine. (B) Binding curves and apparent K d values for E. coli Gpt, Hpt, and Apt binding pppGpp and ppGpp (2 nM [each]). The average values for bound fractions and standard errors of the means (SEM) determined for at least three replicates were plotted and the curve-fitted and K d values determined as previously described ( 37 ). The apparent K d values corresponding to each protein-ligand interaction are shown. (C) Competition assay of Gpt, Hpt, and Apt (20 μM [each]) binding [α- 32 P]ppGpp (2 nM) in the presence of cold competitors (100 μM). The average values for bound fractions and standard errors of the means (SEM) determined for at least three replicates were plotted. Representative DRaCALA spots are shown above the respective diagrams.

Techniques Used: Binding Assay, Competitive Binding Assay

2) Product Images from "Identification and Characterization of Phosphorylation Sites within the Pregnane X Receptor Protein"

Article Title: Identification and Characterization of Phosphorylation Sites within the Pregnane X Receptor Protein

Journal: Biochemical pharmacology

doi: 10.1016/j.bcp.2013.10.015

PXR is phosphorylated in vitro and in cells (A) His-PXR (1 or 2.5 µg) was incubated at 37°C for 30 min with Cdk2 and cyclin E along with [γ- 32 P]-ATP. Samples were resolved on a 4–12% gradient gel, and [γ- 32 P]-ATP incorporation was visualized using a phosphor screen (upper panel), and protein amounts in the samples were detected by SimplyBlue staining of the gel (lower panel). Histone H1 and His-tag were used as a positive and negative substrate control, respectively. The PXR band was indicated with an arrow. (B) Phosphorylation sites identified by using mass spectrometry analysis in His-PXR WT phosphorylated by Cdk2/cyclin E in vitro , and in Flag-PXR WT, Flag-PXR T133A, or Flag-PXR T135A immunoprecipitated from HEK293T cells transiently transfected with corresponding plasmid ( in vivo ). Serine or threonine residues followed by an asterisk (*) indicate phosphorylated residues; UM = unmodified peptide; M = phosphorylated peptide; nd = not detected; nt = not tested. Signal intensities are calculated from area under the curve for the detected precursor ions. (C) Anti-Flag immunoprecipitated samples prepared from HEK293T cells transiently overexpressing either Flag-PXR WT (lanes 1 2) or mutants Flag-PXR T133A (lanes 4 5) or Flag-PXR T135A (lanes 7 8) were resolved on gradient gel and stained using Sypro Ruby stain. (D) Modified peptide sequence TFDTTFS*HFK (asterisk indicating serine phosphorylation), was identified based on assignment of multiple product ions ( b and y ions) in the MS/MS scan of the precursor ion at M/z 665.78. The phosphorylation of serine 167 was confirmed based on the assignment of characteristic “ y-H 3 PO 4 ” ions and other ions (based on a mass loss of 97.9769 Da). (E) Extracted-ion chromatography (XIC) of wild type and mutant PXR sequences showing elution times and signal intensities for the non-modified peptide as well as the singly phosphorylated peptide. Panel (a) and (b) are derived from the immunoprecipitated T133A sample and show the TGAQPLGVQGLTEEQR and T*GAQPLGVQGLTEEQR, respectively. Panel (c) and (d) are derived from the immunoprecipitated T135A sample and show the AGTQPLGVQGLTEEQR and AGT*QPLGVQGLTEEQR, respectively. Panel (e) and (f) are derived from the immunoprecipitated PXR WT sample and show the TGTQPLGVQGLTEEQR and T*GTQPLGVQGLTEEQR/ TGT*QPLGVQGLTEEQR, respectively. Relative abundance (RA) of the signals of the corresponding peptides is noted for each XIC.
Figure Legend Snippet: PXR is phosphorylated in vitro and in cells (A) His-PXR (1 or 2.5 µg) was incubated at 37°C for 30 min with Cdk2 and cyclin E along with [γ- 32 P]-ATP. Samples were resolved on a 4–12% gradient gel, and [γ- 32 P]-ATP incorporation was visualized using a phosphor screen (upper panel), and protein amounts in the samples were detected by SimplyBlue staining of the gel (lower panel). Histone H1 and His-tag were used as a positive and negative substrate control, respectively. The PXR band was indicated with an arrow. (B) Phosphorylation sites identified by using mass spectrometry analysis in His-PXR WT phosphorylated by Cdk2/cyclin E in vitro , and in Flag-PXR WT, Flag-PXR T133A, or Flag-PXR T135A immunoprecipitated from HEK293T cells transiently transfected with corresponding plasmid ( in vivo ). Serine or threonine residues followed by an asterisk (*) indicate phosphorylated residues; UM = unmodified peptide; M = phosphorylated peptide; nd = not detected; nt = not tested. Signal intensities are calculated from area under the curve for the detected precursor ions. (C) Anti-Flag immunoprecipitated samples prepared from HEK293T cells transiently overexpressing either Flag-PXR WT (lanes 1 2) or mutants Flag-PXR T133A (lanes 4 5) or Flag-PXR T135A (lanes 7 8) were resolved on gradient gel and stained using Sypro Ruby stain. (D) Modified peptide sequence TFDTTFS*HFK (asterisk indicating serine phosphorylation), was identified based on assignment of multiple product ions ( b and y ions) in the MS/MS scan of the precursor ion at M/z 665.78. The phosphorylation of serine 167 was confirmed based on the assignment of characteristic “ y-H 3 PO 4 ” ions and other ions (based on a mass loss of 97.9769 Da). (E) Extracted-ion chromatography (XIC) of wild type and mutant PXR sequences showing elution times and signal intensities for the non-modified peptide as well as the singly phosphorylated peptide. Panel (a) and (b) are derived from the immunoprecipitated T133A sample and show the TGAQPLGVQGLTEEQR and T*GAQPLGVQGLTEEQR, respectively. Panel (c) and (d) are derived from the immunoprecipitated T135A sample and show the AGTQPLGVQGLTEEQR and AGT*QPLGVQGLTEEQR, respectively. Panel (e) and (f) are derived from the immunoprecipitated PXR WT sample and show the TGTQPLGVQGLTEEQR and T*GTQPLGVQGLTEEQR/ TGT*QPLGVQGLTEEQR, respectively. Relative abundance (RA) of the signals of the corresponding peptides is noted for each XIC.

Techniques Used: In Vitro, Incubation, Staining, Mass Spectrometry, Immunoprecipitation, Transfection, Plasmid Preparation, In Vivo, Modification, Sequencing, Ion Chromatography, Mutagenesis, Derivative Assay

3) Product Images from "CCR4 and CAF1 deadenylases have an intrinsic activity to remove the post-poly(A) sequence"

Article Title: CCR4 and CAF1 deadenylases have an intrinsic activity to remove the post-poly(A) sequence

Journal: RNA

doi: 10.1261/rna.057679.116

The balance between the poly(A) length and the downstream post-poly(A) length affects deadenylation efficiency. ( A ) Schematic representation of a series of Mini- let-7 reporter constructs. These reporter RNAs include four let-7 target sites and were capped with [α- 32 P] GTP. The subscripts show the length of an RNA sequence including the shown base. The asterisks indicate a radiolabel. ( B ) Deadenylation assay for Mini- let-7 reporter RNAs with S2 cell lysate overexpressed Ago1. ( C ) The signal intensity of the bands in B was quantified, and the ratio of the intensity from the lower band for the total intensity was calculated and plotted. Mini- let-7 -A 20 effectively shifted to A 0 . While the length of the post-poly(A) sequence negatively correlated with the deadenylation efficiency, elongating the internal poly(A) sequence improved. The graph shows means and standard deviations ( n = 3). ( D ) Deadenylation assay for Mini- let-7 -A 60 C 10 and Mini- let-7 -N 40 A 20 C 10 with S2 cell lysate overexpressed Ago1. ( E ) The signal intensity of the bands in D was quantified, and the ratio of the intensity from the lower band for the total intensity was calculated and plotted. Mini- let-7 -A 60 C 10 was deadenylated more effectively than Mini- let-7 -N 40 A 20 C 10 . The graph shows means and standard deviations ( n = 3).
Figure Legend Snippet: The balance between the poly(A) length and the downstream post-poly(A) length affects deadenylation efficiency. ( A ) Schematic representation of a series of Mini- let-7 reporter constructs. These reporter RNAs include four let-7 target sites and were capped with [α- 32 P] GTP. The subscripts show the length of an RNA sequence including the shown base. The asterisks indicate a radiolabel. ( B ) Deadenylation assay for Mini- let-7 reporter RNAs with S2 cell lysate overexpressed Ago1. ( C ) The signal intensity of the bands in B was quantified, and the ratio of the intensity from the lower band for the total intensity was calculated and plotted. Mini- let-7 -A 20 effectively shifted to A 0 . While the length of the post-poly(A) sequence negatively correlated with the deadenylation efficiency, elongating the internal poly(A) sequence improved. The graph shows means and standard deviations ( n = 3). ( D ) Deadenylation assay for Mini- let-7 -A 60 C 10 and Mini- let-7 -N 40 A 20 C 10 with S2 cell lysate overexpressed Ago1. ( E ) The signal intensity of the bands in D was quantified, and the ratio of the intensity from the lower band for the total intensity was calculated and plotted. Mini- let-7 -A 60 C 10 was deadenylated more effectively than Mini- let-7 -N 40 A 20 C 10 . The graph shows means and standard deviations ( n = 3).

Techniques Used: Construct, Sequencing

4) Product Images from "Practical and general synthesis of 5?-adenylated RNA (5?-AppRNA)"

Article Title: Practical and general synthesis of 5?-adenylated RNA (5?-AppRNA)

Journal: RNA

doi: 10.1261/rna.5247704

5′-adenylation of long RNA substrates. ( A ) Schematic diagram of the experimental strategy. The > 100-mer RNA substrate is too long for 5′-AppRNA formation to induce a measurable gel shift relative to a 5′-monophosphate. Therefore, an appropriate 8–17 deoxyribozyme is used to cleave the 5′-portion of the RNA substrate, leaving a small fragment for which 5′-AppRNA formation does cause a gel shift. ( B ) The strategy in A applied to the 160-nt P4–P6 domain of the Tetrahymena group I intron RNA. Blocking oligos were uncapped. The three time points are at 0.5 min, 10 min, and 1 h (6% PAGE). The RNA substrate was internally radiolabeled by transcription incorporating α- 32 P-ATP; the 5′-monophosphate was provided by performing the transcription in the presence of excess GMP (see Materials and Methods). Although the side products have not been studied in great detail, the side product formed in the first experiment (P4–P6 with no DNA blocking oligo) is tentatively assigned as circularized P4–P6 on the basis of attempted 5′- 32 P-radiolabeling with T4 polynucleotide kinase and γ- 32 P-ATP; no reaction was observed alongside a positive control. Only the lower band (a mixture of 5′-monophosphate and 5′-AppRNA) was carried to the 8–17 deoxyribozyme cleavage experiment. std, P4–P6 standard RNA carried through all reactions with no blocking oligo, except that T4 RNA ligase was omitted. ( C ) The strategy in A ).
Figure Legend Snippet: 5′-adenylation of long RNA substrates. ( A ) Schematic diagram of the experimental strategy. The > 100-mer RNA substrate is too long for 5′-AppRNA formation to induce a measurable gel shift relative to a 5′-monophosphate. Therefore, an appropriate 8–17 deoxyribozyme is used to cleave the 5′-portion of the RNA substrate, leaving a small fragment for which 5′-AppRNA formation does cause a gel shift. ( B ) The strategy in A applied to the 160-nt P4–P6 domain of the Tetrahymena group I intron RNA. Blocking oligos were uncapped. The three time points are at 0.5 min, 10 min, and 1 h (6% PAGE). The RNA substrate was internally radiolabeled by transcription incorporating α- 32 P-ATP; the 5′-monophosphate was provided by performing the transcription in the presence of excess GMP (see Materials and Methods). Although the side products have not been studied in great detail, the side product formed in the first experiment (P4–P6 with no DNA blocking oligo) is tentatively assigned as circularized P4–P6 on the basis of attempted 5′- 32 P-radiolabeling with T4 polynucleotide kinase and γ- 32 P-ATP; no reaction was observed alongside a positive control. Only the lower band (a mixture of 5′-monophosphate and 5′-AppRNA) was carried to the 8–17 deoxyribozyme cleavage experiment. std, P4–P6 standard RNA carried through all reactions with no blocking oligo, except that T4 RNA ligase was omitted. ( C ) The strategy in A ).

Techniques Used: Electrophoretic Mobility Shift Assay, Blocking Assay, Polyacrylamide Gel Electrophoresis, Radioactivity, Positive Control

5) Product Images from "?-Arrestins Aly1 and Aly2 Regulate Intracellular Trafficking in Response to Nutrient Signaling"

Article Title: ?-Arrestins Aly1 and Aly2 Regulate Intracellular Trafficking in Response to Nutrient Signaling

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E10-07-0636

Aly2 interacts with and requires Npr1 to promote Gap1 PM-localization. (A) BJ5459 or BJ5459-Npr1-MYC cells expressing GST (pKK212), GST-Aly1 (pKK212-Aly1), or GST-Aly2 (pKK212-Aly2) were grown in SC-0.25% NH 4 . Protein extracts were split, with half used for GST and half for anti-MYC Ab purifications, and copurification assessed by WB. Samples were run on one gel, but line denotes lane removal. (B) WT (BY4741) or npr1 Δ (2029) cells with pRS425, -Aly1 or -Aly2 were grown in MIN-0.25% NH 4 , washed, and inoculated at equal density into either MIN-0.1% GLN or MIN-0.1% citrulline (CIT). Growth was monitored using OD 600 readings, taken every 30 min with a Tecan Genios microtiter plate reader. (C) Growth of WT (BY4741) or npr1 Δ (2029) cells with pRS425, -Aly1, or -Aly2 on MIN-0.5% NH 4 ± AzC. (D) Prototrophic WT (BY4741) and npr1 Δ (2029) with pCK283 and pRS426, - ALY1 , or - ALY2 were assayed for [ 14 C]citrulline uptake. The mean uptake rate ± SDM for three replicates is shown as % relative to WT. (E and F) Prototrophic npr1 ΔΔ (32029) cells with Gap1-GFP (pCK230), pRS313 and pRS425, -Aly1, or -Aly2 were grown in SC-0.5% NH 4 , washed, and grown for 3 h in MIN-0.5% NH 4 and (E) cell extracts were assessed by WB or (F) Gap1-GFP was visualized using fluorescence microscopy (scale bar, 5 μm). (G) GST-Aly1 (pKK212-Aly1) or -Aly2 (pKK212-Aly2) were purified from extracts of WT (BJ5459) or npr1 Δ (BJ5459- npr1 Δ:: KanMX ) cells grown in SC-0.25% NH 4 and assessed by WB. Similar results were obtained using GFP-Aly1 and -Aly2 extracted from WT (BY4741) or npr1 Δ (2029) cells (data not shown). Phosphorylation of GST-Aly2 was further analyzed using mock (−) or lambda phosphatase treatment (λ-PP). (H) pET and pET-Aly2 were purified from E. coli and incubated with [γ- 32 P]ATP kinase cocktail in the presence (+) or absence (−) of Npr1. Proteins were analyzed by SDS-PAGE and imaged on a Typhoon scanner for 32 P quantification or stained for total protein. pET-Aly2 phosphorylation ± Npr1 is shown (left-hand portion of panel). The mean fold-increase in phospho-signal upon addition of Npr1 kinase (normalized for loading) is plotted from three replicate experiments ± SDM for both pET-Aly2 and the pET tag alone (the latter is not phosphorylated by Npr1) in the right-hand portion of the panel.
Figure Legend Snippet: Aly2 interacts with and requires Npr1 to promote Gap1 PM-localization. (A) BJ5459 or BJ5459-Npr1-MYC cells expressing GST (pKK212), GST-Aly1 (pKK212-Aly1), or GST-Aly2 (pKK212-Aly2) were grown in SC-0.25% NH 4 . Protein extracts were split, with half used for GST and half for anti-MYC Ab purifications, and copurification assessed by WB. Samples were run on one gel, but line denotes lane removal. (B) WT (BY4741) or npr1 Δ (2029) cells with pRS425, -Aly1 or -Aly2 were grown in MIN-0.25% NH 4 , washed, and inoculated at equal density into either MIN-0.1% GLN or MIN-0.1% citrulline (CIT). Growth was monitored using OD 600 readings, taken every 30 min with a Tecan Genios microtiter plate reader. (C) Growth of WT (BY4741) or npr1 Δ (2029) cells with pRS425, -Aly1, or -Aly2 on MIN-0.5% NH 4 ± AzC. (D) Prototrophic WT (BY4741) and npr1 Δ (2029) with pCK283 and pRS426, - ALY1 , or - ALY2 were assayed for [ 14 C]citrulline uptake. The mean uptake rate ± SDM for three replicates is shown as % relative to WT. (E and F) Prototrophic npr1 ΔΔ (32029) cells with Gap1-GFP (pCK230), pRS313 and pRS425, -Aly1, or -Aly2 were grown in SC-0.5% NH 4 , washed, and grown for 3 h in MIN-0.5% NH 4 and (E) cell extracts were assessed by WB or (F) Gap1-GFP was visualized using fluorescence microscopy (scale bar, 5 μm). (G) GST-Aly1 (pKK212-Aly1) or -Aly2 (pKK212-Aly2) were purified from extracts of WT (BJ5459) or npr1 Δ (BJ5459- npr1 Δ:: KanMX ) cells grown in SC-0.25% NH 4 and assessed by WB. Similar results were obtained using GFP-Aly1 and -Aly2 extracted from WT (BY4741) or npr1 Δ (2029) cells (data not shown). Phosphorylation of GST-Aly2 was further analyzed using mock (−) or lambda phosphatase treatment (λ-PP). (H) pET and pET-Aly2 were purified from E. coli and incubated with [γ- 32 P]ATP kinase cocktail in the presence (+) or absence (−) of Npr1. Proteins were analyzed by SDS-PAGE and imaged on a Typhoon scanner for 32 P quantification or stained for total protein. pET-Aly2 phosphorylation ± Npr1 is shown (left-hand portion of panel). The mean fold-increase in phospho-signal upon addition of Npr1 kinase (normalized for loading) is plotted from three replicate experiments ± SDM for both pET-Aly2 and the pET tag alone (the latter is not phosphorylated by Npr1) in the right-hand portion of the panel.

Techniques Used: Expressing, Copurification, Western Blot, Fluorescence, Microscopy, Purification, Positron Emission Tomography, Incubation, SDS Page, Staining

6) Product Images from "LOV Histidine Kinase Modulates the General Stress Response System and Affects the virB Operon Expression in Brucella abortus"

Article Title: LOV Histidine Kinase Modulates the General Stress Response System and Affects the virB Operon Expression in Brucella abortus

Journal: PLoS ONE

doi: 10.1371/journal.pone.0124058

Phosphotransfer reaction between Brucella LOVHK and RRs. Purified LOVHK protein was illuminated in phosphorylation buffer containing [γ- 32 P] ATP. After 15 min at 37°C, purified response regulators were added to the mixture to a final concentration of 2.5 μM each for the three proteins. The final concentration of LOVHK was also 2.5 μM. At the indicated times after addition of the corresponding response regulators, aliquots were drawn and separated by 15% SDS-PAGE. Autoradiograms are shown on the left, and the graphs on the right side indicate the relative intensity of each band to the total intensity at time 20 seconds. The experiment was repeated three times, and a representative experiment is shown. Numbers above the autoradiograms indicate the time in seconds (columns 1 and 2) or in minutes (columns from 3 to 9) respectively. A. Phosphotransfer between LOVHK and PhyR. B. Phosphotransfer between LOVHK and LovR. C. Phosphotransfer between LOVHK, PhyR and LovR simultaneously. LOVHK: blue circles, PhyR: green triangles, LovR: red rhomboids, total intensity: black squares. Molecular weights of protein constructions are indicated in Fig 1A .
Figure Legend Snippet: Phosphotransfer reaction between Brucella LOVHK and RRs. Purified LOVHK protein was illuminated in phosphorylation buffer containing [γ- 32 P] ATP. After 15 min at 37°C, purified response regulators were added to the mixture to a final concentration of 2.5 μM each for the three proteins. The final concentration of LOVHK was also 2.5 μM. At the indicated times after addition of the corresponding response regulators, aliquots were drawn and separated by 15% SDS-PAGE. Autoradiograms are shown on the left, and the graphs on the right side indicate the relative intensity of each band to the total intensity at time 20 seconds. The experiment was repeated three times, and a representative experiment is shown. Numbers above the autoradiograms indicate the time in seconds (columns 1 and 2) or in minutes (columns from 3 to 9) respectively. A. Phosphotransfer between LOVHK and PhyR. B. Phosphotransfer between LOVHK and LovR. C. Phosphotransfer between LOVHK, PhyR and LovR simultaneously. LOVHK: blue circles, PhyR: green triangles, LovR: red rhomboids, total intensity: black squares. Molecular weights of protein constructions are indicated in Fig 1A .

Techniques Used: Purification, Concentration Assay, SDS Page

7) Product Images from "Plk4-dependent phosphorylation of STIL is required for centriole duplication"

Article Title: Plk4-dependent phosphorylation of STIL is required for centriole duplication

Journal: Biology Open

doi: 10.1242/bio.201411023

Phosphorylation of STIL by Plk4 triggers centriole duplication. (A) Flag-STIL full-length or 5A mutant (S871A/S873A/S874A/S1116A/T1250A) expressed in HEK293T cells and immunoprecipitated with anti-Flag antibodies was incubated with bacterially expressed Zz-Plk4 in the presence of [γ- 32 P]-ATP. In vitro kinase assay with Flag-STIL or Plk4 alone served as a control. Kinase assays were analyzed by SDS-PAGE, Coomassie Blue staining and autoradiography. (B) Co immunoprecipitation of Flag-STIL wt/5A and Myc-Plk4. Lysates from HEK293T cells transfected with the indicated plasmids were subjected to immunoprecipitation using anti-Flag antibodies. Input and IP samples were analyzed by western blotting with antibodies against Flag- and Myc-tag. The asterisk marks an unspecific band recognized by the anti-Myc antibody. The dividing lane indicates grouping of images from different parts of the same gel, as an intervening lane was removed for presentation purposes. (C) U2OS cells transiently expressing Flag EV, Flag-STIL wt or Flag-STIL 5A were analyzed by indirect immunofluorescence using staining with anti-CP110 and mouse anti-Flag antibodies 72 h after transfection. The number of transfected cells with more than four centrioles was determined based on CP110 staining. Values in the graph are mean percentages±s.d. from three independent experiments, 50 transfected cells were analyzed in each experiment (***P
Figure Legend Snippet: Phosphorylation of STIL by Plk4 triggers centriole duplication. (A) Flag-STIL full-length or 5A mutant (S871A/S873A/S874A/S1116A/T1250A) expressed in HEK293T cells and immunoprecipitated with anti-Flag antibodies was incubated with bacterially expressed Zz-Plk4 in the presence of [γ- 32 P]-ATP. In vitro kinase assay with Flag-STIL or Plk4 alone served as a control. Kinase assays were analyzed by SDS-PAGE, Coomassie Blue staining and autoradiography. (B) Co immunoprecipitation of Flag-STIL wt/5A and Myc-Plk4. Lysates from HEK293T cells transfected with the indicated plasmids were subjected to immunoprecipitation using anti-Flag antibodies. Input and IP samples were analyzed by western blotting with antibodies against Flag- and Myc-tag. The asterisk marks an unspecific band recognized by the anti-Myc antibody. The dividing lane indicates grouping of images from different parts of the same gel, as an intervening lane was removed for presentation purposes. (C) U2OS cells transiently expressing Flag EV, Flag-STIL wt or Flag-STIL 5A were analyzed by indirect immunofluorescence using staining with anti-CP110 and mouse anti-Flag antibodies 72 h after transfection. The number of transfected cells with more than four centrioles was determined based on CP110 staining. Values in the graph are mean percentages±s.d. from three independent experiments, 50 transfected cells were analyzed in each experiment (***P

Techniques Used: Mutagenesis, Immunoprecipitation, Incubation, In Vitro, Kinase Assay, SDS Page, Staining, Autoradiography, Transfection, Western Blot, Expressing, Immunofluorescence

Phosphorylation of STIL by Plk4. (A) Full-length Flag-STIL expressed in HEK293T cells and immunoprecipitated with anti-Flag antibodies was incubated with bacterially expressed Zz-Plk4 in the presence of [γ- 32 P]-ATP. In vitro kinase assay with Flag-STIL or Plk4 alone served as a control. Kinase assays were analyzed by SDS-PAGE, Coomassie Blue staining and autoradiography. (B) Indicated Flag-STIL fragments were expressed in HEK293T cells and immunoprecipitated with anti-Flag antibodies. Immunoprecipitation fractions were incubated with bacterially expressed Zz-Plk4 in the presence of [γ- 32 P]-ATP, followed by SDS-PAGE and autoradiography. In vitro kinase assay with Flag-STIL fragments or Plk4 alone is shown as control. The asterisk indicates phosphorylated Flag-STIL 781-1287. 10% of each precipitation fraction was analyzed by western blotting using anti-Plk4 and anti-Flag antibodies. (C) Plk4 phosphorylation sites in the STIL protein identified by mass spectrometry analysis of bacterially purified GST-STIL 1-619 and 619-1287 phosphorylated in vitro by Zz-Plk4. Alignment of the identified sites in human, mouse, Xenopus and zebrafish STIL and Drosophila Ana2 is shown.
Figure Legend Snippet: Phosphorylation of STIL by Plk4. (A) Full-length Flag-STIL expressed in HEK293T cells and immunoprecipitated with anti-Flag antibodies was incubated with bacterially expressed Zz-Plk4 in the presence of [γ- 32 P]-ATP. In vitro kinase assay with Flag-STIL or Plk4 alone served as a control. Kinase assays were analyzed by SDS-PAGE, Coomassie Blue staining and autoradiography. (B) Indicated Flag-STIL fragments were expressed in HEK293T cells and immunoprecipitated with anti-Flag antibodies. Immunoprecipitation fractions were incubated with bacterially expressed Zz-Plk4 in the presence of [γ- 32 P]-ATP, followed by SDS-PAGE and autoradiography. In vitro kinase assay with Flag-STIL fragments or Plk4 alone is shown as control. The asterisk indicates phosphorylated Flag-STIL 781-1287. 10% of each precipitation fraction was analyzed by western blotting using anti-Plk4 and anti-Flag antibodies. (C) Plk4 phosphorylation sites in the STIL protein identified by mass spectrometry analysis of bacterially purified GST-STIL 1-619 and 619-1287 phosphorylated in vitro by Zz-Plk4. Alignment of the identified sites in human, mouse, Xenopus and zebrafish STIL and Drosophila Ana2 is shown.

Techniques Used: Immunoprecipitation, Incubation, In Vitro, Kinase Assay, SDS Page, Staining, Autoradiography, Western Blot, Mass Spectrometry, Purification

8) Product Images from "Viral Mimicry of Cdc2/Cyclin-Dependent Kinase 1 Mediates Disruption of Nuclear Lamina during Human Cytomegalovirus Nuclear Egress"

Article Title: Viral Mimicry of Cdc2/Cyclin-Dependent Kinase 1 Mediates Disruption of Nuclear Lamina during Human Cytomegalovirus Nuclear Egress

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1000275

In vitro phosphorylation of lamin A by GST-UL97. (A) Recombinant His-tagged lamin A was incubated in kinase reaction buffer in the presence of γ- 32 P-ATP either alone (no kinase), with catalytically deficient GST-UL97 K355Q (K355Q), or with wild-type GST-UL97 (GST97 WT). GST-UL97 K335Q or wild-type GST-UL97 were also incubated in kinase buffer without lamin A. Following termination of kinase reactions, proteins were resolved by SDS-PAGE. Signal from incorporation of 32 P was detected by exposure to a phosphorscreen (top panel), and total protein was detected by Coomassie brilliant blue staining (bottom panel). The positions of radiolabeled GST-UL97 (GST97) and lamin A, and Coomassie stained lamin A are indicated. (The amounts of GST-UL97 were too small to see on the stained gel.) (B) UL97 autophosphorylation and labeling of lamin A were quantified following in in vitro kinase reactions in the presence of varying concentrations of maribavir (MBV). Signal detected from 32 P incorporation for autophosphorylation of GST-UL97 and phosphorylation of His-tagged lamin A are plotted as a percent of the signal detected in the absence of drug. The results taken together show that UL97 phosphorylates lamin A in vitro.
Figure Legend Snippet: In vitro phosphorylation of lamin A by GST-UL97. (A) Recombinant His-tagged lamin A was incubated in kinase reaction buffer in the presence of γ- 32 P-ATP either alone (no kinase), with catalytically deficient GST-UL97 K355Q (K355Q), or with wild-type GST-UL97 (GST97 WT). GST-UL97 K335Q or wild-type GST-UL97 were also incubated in kinase buffer without lamin A. Following termination of kinase reactions, proteins were resolved by SDS-PAGE. Signal from incorporation of 32 P was detected by exposure to a phosphorscreen (top panel), and total protein was detected by Coomassie brilliant blue staining (bottom panel). The positions of radiolabeled GST-UL97 (GST97) and lamin A, and Coomassie stained lamin A are indicated. (The amounts of GST-UL97 were too small to see on the stained gel.) (B) UL97 autophosphorylation and labeling of lamin A were quantified following in in vitro kinase reactions in the presence of varying concentrations of maribavir (MBV). Signal detected from 32 P incorporation for autophosphorylation of GST-UL97 and phosphorylation of His-tagged lamin A are plotted as a percent of the signal detected in the absence of drug. The results taken together show that UL97 phosphorylates lamin A in vitro.

Techniques Used: In Vitro, Recombinant, Incubation, SDS Page, Staining, Labeling

9) Product Images from "Physicochemical analysis of rotavirus segment 11 supports a 'modified panhandle' structure and not the predicted alternative tRNA-like structure (TRLS)"

Article Title: Physicochemical analysis of rotavirus segment 11 supports a 'modified panhandle' structure and not the predicted alternative tRNA-like structure (TRLS)

Journal: Archives of Virology

doi: 10.1007/s00705-013-1802-8

RNase T1 cleavage results of single-stranded guanines in the 5’-terminal sequence of rotavirus RNA11. Single-stranded rotavirus RNA11 was labelled at the 5’ terminus using [γ- 32 P]ATP, subjected to partial digestion by RNase T1, and resolved on a 12 % polyacrylamide 7 M urea gel. The dark bands in the last column on the right show the positions of the single-stranded guanines cleaved by RNase T1, compared to the third column from the left (no-enzyme control). The first and second columns from the left represent an RNA11 sequence ladder generated from the same RNA by alkaline hydrolysis for 5 and 10 min, respectively
Figure Legend Snippet: RNase T1 cleavage results of single-stranded guanines in the 5’-terminal sequence of rotavirus RNA11. Single-stranded rotavirus RNA11 was labelled at the 5’ terminus using [γ- 32 P]ATP, subjected to partial digestion by RNase T1, and resolved on a 12 % polyacrylamide 7 M urea gel. The dark bands in the last column on the right show the positions of the single-stranded guanines cleaved by RNase T1, compared to the third column from the left (no-enzyme control). The first and second columns from the left represent an RNA11 sequence ladder generated from the same RNA by alkaline hydrolysis for 5 and 10 min, respectively

Techniques Used: Sequencing, Generated

10) Product Images from "Impact of an invasive nitrogen-fixing tree on arbuscular mycorrhizal fungi and the development of native species"

Article Title: Impact of an invasive nitrogen-fixing tree on arbuscular mycorrhizal fungi and the development of native species

Journal: AoB Plants

doi: 10.1093/aobpla/plw018

Percentage of colonization in P. lanceolata roots. NSA, non-sterilized acacia roots; SA, sterilized acacia roots; NSS, non-sterilized shrub roots and SS, sterilized shrub roots. Different letters indicate significant differences at P ≤ 0.05 level.
Figure Legend Snippet: Percentage of colonization in P. lanceolata roots. NSA, non-sterilized acacia roots; SA, sterilized acacia roots; NSS, non-sterilized shrub roots and SS, sterilized shrub roots. Different letters indicate significant differences at P ≤ 0.05 level.

Techniques Used:

11) Product Images from "Impact of an invasive nitrogen-fixing tree on arbuscular mycorrhizal fungi and the development of native species"

Article Title: Impact of an invasive nitrogen-fixing tree on arbuscular mycorrhizal fungi and the development of native species

Journal: AoB Plants

doi: 10.1093/aobpla/plw018

Percentage of colonization in P. lanceolata roots. NSA, non-sterilized acacia roots; SA, sterilized acacia roots; NSS, non-sterilized shrub roots and SS, sterilized shrub roots. Different letters indicate significant differences at P ≤ 0.05 level.
Figure Legend Snippet: Percentage of colonization in P. lanceolata roots. NSA, non-sterilized acacia roots; SA, sterilized acacia roots; NSS, non-sterilized shrub roots and SS, sterilized shrub roots. Different letters indicate significant differences at P ≤ 0.05 level.

Techniques Used:

12) Product Images from "Demonstration of Phosphoryl Group Transfer Indicates That the ATP-binding Cassette (ABC) Transporter Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Exhibits Adenylate Kinase Activity *"

Article Title: Demonstration of Phosphoryl Group Transfer Indicates That the ATP-binding Cassette (ABC) Transporter Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Exhibits Adenylate Kinase Activity *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M112.408450

Membrane-inserted CFTR catalyzes phosphotransfer from [γ- 32 P]GTP to N 3 -AMP. A , Western blot ( WB ) probed with antibody 13-1. Letters label highly ( C ) and core glycosylated ( B ) CFTR. Each lane represents 30 μg of membrane protein. B , autoradiograph and Western blot (probed with antibody M3A7) of the same gel. Experiments were performed as illustrated in Fig. 1 . Experimental conditions are indicated below the lanes. N 3 -AMP concentration was 65 μ m . Comparing the autoradiograph and Western blot corroborated that the labeled band was CFTR. C , CFTR photolabeling with 8-N 3 -AMP and 2-N 3 -AMP. N 3 -AMP concentration was 65 μ m . To compare the results from different autoradiographs, data were normalized to CFTR radioactivity under conditions indicated below bar 4. Asterisks indicate p ≤ 0.001 when compared with bar 4, and double daggers indicate p ≤ 0.001 when compared with bar 3 (one-way analysis of variance followed by the Holm-Sidak method for multiple comparisons, n = 3).
Figure Legend Snippet: Membrane-inserted CFTR catalyzes phosphotransfer from [γ- 32 P]GTP to N 3 -AMP. A , Western blot ( WB ) probed with antibody 13-1. Letters label highly ( C ) and core glycosylated ( B ) CFTR. Each lane represents 30 μg of membrane protein. B , autoradiograph and Western blot (probed with antibody M3A7) of the same gel. Experiments were performed as illustrated in Fig. 1 . Experimental conditions are indicated below the lanes. N 3 -AMP concentration was 65 μ m . Comparing the autoradiograph and Western blot corroborated that the labeled band was CFTR. C , CFTR photolabeling with 8-N 3 -AMP and 2-N 3 -AMP. N 3 -AMP concentration was 65 μ m . To compare the results from different autoradiographs, data were normalized to CFTR radioactivity under conditions indicated below bar 4. Asterisks indicate p ≤ 0.001 when compared with bar 4, and double daggers indicate p ≤ 0.001 when compared with bar 3 (one-way analysis of variance followed by the Holm-Sidak method for multiple comparisons, n = 3).

Techniques Used: Western Blot, Autoradiography, Concentration Assay, Labeling, Radioactivity

Model of CFTR labeling through phosphoryl group transfer between [γ- 32 P]GTP and N 3 -AMP followed by UV-mediated cross-linking of the resulting N 3 -[β- 32 P]ADP and solubilization and immunoprecipitation ( IP ) of CFTR. P * indicates a radioactive phosphoryl group containing 32 P. In each NBD, the open rectangle represents the Walker A motif, and the open triangle represents the signature motif. The binding site for AMP is not known.
Figure Legend Snippet: Model of CFTR labeling through phosphoryl group transfer between [γ- 32 P]GTP and N 3 -AMP followed by UV-mediated cross-linking of the resulting N 3 -[β- 32 P]ADP and solubilization and immunoprecipitation ( IP ) of CFTR. P * indicates a radioactive phosphoryl group containing 32 P. In each NBD, the open rectangle represents the Walker A motif, and the open triangle represents the signature motif. The binding site for AMP is not known.

Techniques Used: Labeling, Immunoprecipitation, Binding Assay

CFTR has intrinsic adenylate kinase activity. A , autoradiograph of immunoprecipitated CFTR fractionated on a 6% SDS-polyacrylamide gel. Experiments were performed as illustrated in Fig. 1 . Membranes containing 30 μg of protein from CFTR-expressing HeLa cells ( lanes 3–5 ) or control membranes ( contr. membr. ) containing 30 μg of protein from HeLa cells not expressing recombinant CFTR ( lane 1 ) were used. In lane 6 , membranes containing 90 μg of protein from S1248F CFTR-expressing HeLa cells were employed. Membranes were incubated together with 50 μ m 2-N 3 -AMP and 30 μCi of [γ- 32 P]GTP (6000 Ci/mmol) for 5 min at 37 °C followed by UV irradiation for 30 s (302 nm, 8-watt lamp) at a distance of 5 cm as described under “Experimental Procedures.” The sample of lane 4 was not UV-irradiated. In lane 2 , 30 μg of membranes from HeLa cells not expressing recombinant CFTR (control membranes) were incubated with 50 μ m 2-N 3 -AMP and 30 μCi of [γ- 32 P]GTP (6000 Ci/mmol) for 5 min at 37 °C. Then 30 μg of membranes containing CFTR were added on ice before UV irradiation (30 s, 302 nm, 8-watt lamp). In all cases, CFTR was then solubilized and immunoprecipitated as described under “Experimental Procedures.” B , summary data. Radioactivity incorporated into CFTR was normalized to radioactivity for conditions indicated below bar 5. Asterisks indicate p = 0.029 when compared with bar 5 (Mann-Whitney rank sum test, n = 4). No significant differences were detected between bars 1–4 and 6 (Kruskal-Wallis one-way analysis of variance on ranks, n = 4). C , Western blot probed with CFTR antibody 13-1. 30 μg (control membranes and membranes with wild-type CFTR, lanes 1–3 ) and 90 μg (membranes with S1248F CFTR, lane 4 ) of protein were used.
Figure Legend Snippet: CFTR has intrinsic adenylate kinase activity. A , autoradiograph of immunoprecipitated CFTR fractionated on a 6% SDS-polyacrylamide gel. Experiments were performed as illustrated in Fig. 1 . Membranes containing 30 μg of protein from CFTR-expressing HeLa cells ( lanes 3–5 ) or control membranes ( contr. membr. ) containing 30 μg of protein from HeLa cells not expressing recombinant CFTR ( lane 1 ) were used. In lane 6 , membranes containing 90 μg of protein from S1248F CFTR-expressing HeLa cells were employed. Membranes were incubated together with 50 μ m 2-N 3 -AMP and 30 μCi of [γ- 32 P]GTP (6000 Ci/mmol) for 5 min at 37 °C followed by UV irradiation for 30 s (302 nm, 8-watt lamp) at a distance of 5 cm as described under “Experimental Procedures.” The sample of lane 4 was not UV-irradiated. In lane 2 , 30 μg of membranes from HeLa cells not expressing recombinant CFTR (control membranes) were incubated with 50 μ m 2-N 3 -AMP and 30 μCi of [γ- 32 P]GTP (6000 Ci/mmol) for 5 min at 37 °C. Then 30 μg of membranes containing CFTR were added on ice before UV irradiation (30 s, 302 nm, 8-watt lamp). In all cases, CFTR was then solubilized and immunoprecipitated as described under “Experimental Procedures.” B , summary data. Radioactivity incorporated into CFTR was normalized to radioactivity for conditions indicated below bar 5. Asterisks indicate p = 0.029 when compared with bar 5 (Mann-Whitney rank sum test, n = 4). No significant differences were detected between bars 1–4 and 6 (Kruskal-Wallis one-way analysis of variance on ranks, n = 4). C , Western blot probed with CFTR antibody 13-1. 30 μg (control membranes and membranes with wild-type CFTR, lanes 1–3 ) and 90 μg (membranes with S1248F CFTR, lane 4 ) of protein were used.

Techniques Used: Activity Assay, Autoradiography, Immunoprecipitation, Expressing, Recombinant, Incubation, Irradiation, Radioactivity, MANN-WHITNEY, Western Blot

13) Product Images from "Mechanism of Action of T-705 Ribosyl Triphosphate against Influenza Virus RNA Polymerase"

Article Title: Mechanism of Action of T-705 Ribosyl Triphosphate against Influenza Virus RNA Polymerase

Journal: Antimicrobial Agents and Chemotherapy

doi: 10.1128/AAC.00649-13

Inhibitory activity of T-705RTP versus the incorporation of ATP, GTP, CTP, or UTP. The activity of influenza virus RNA polymerase in the presence of T-705RTP was determined at different concentrations of NTPs. The incorporation of [α- 32 P]GTP was
Figure Legend Snippet: Inhibitory activity of T-705RTP versus the incorporation of ATP, GTP, CTP, or UTP. The activity of influenza virus RNA polymerase in the presence of T-705RTP was determined at different concentrations of NTPs. The incorporation of [α- 32 P]GTP was

Techniques Used: Activity Assay

14) Product Images from "Single-molecule tracking of the transcription cycle by sub-second RNA detection"

Article Title: Single-molecule tracking of the transcription cycle by sub-second RNA detection

Journal: eLife

doi: 10.7554/eLife.01775

Labeling and biochemical characterization of T7 RNAP activity. ( A ) Synthesis of Cy5-conjugated HaloTag Ligand ( S3 ) used to label RNAP from Cy5 acid S1 and HaloTag ligand-amine S2 . See ‘Materials and methods’ for details. ( B ) SDS-PAGE gel images of the unlabeled (‘RNAP’: no HaloTag; ‘Halo-RNAP’: with HaloTag) and the Cy5-labeled Halo-RNAP (‘Cy5-Halo-RNAP’) T7 RNAP. All RNAPs have a 6(His) tag at the N-terminus used for purification. Top: image stained with Coomassie Brilliant Blue. Bottom: the same gel scanned for Cy5 fluorescence (prior to Coomassie staining). ( C ) In vitro transcription activity of RNAP. The concentrations of RNAP derivatives were as indicated. The DNA template (a PCR fragment spanning from −75 to +295 of the concensus T7 RNAP promoter), was used at 10 nM. RNA products were labeled by incorporation of α 32 P-ATP, resolved on a denaturing polyacrylamide gel, and imaged by autoradiography. The major bands at ∼295 nt are the expected run-off products. DOI: http://dx.doi.org/10.7554/eLife.01775.009
Figure Legend Snippet: Labeling and biochemical characterization of T7 RNAP activity. ( A ) Synthesis of Cy5-conjugated HaloTag Ligand ( S3 ) used to label RNAP from Cy5 acid S1 and HaloTag ligand-amine S2 . See ‘Materials and methods’ for details. ( B ) SDS-PAGE gel images of the unlabeled (‘RNAP’: no HaloTag; ‘Halo-RNAP’: with HaloTag) and the Cy5-labeled Halo-RNAP (‘Cy5-Halo-RNAP’) T7 RNAP. All RNAPs have a 6(His) tag at the N-terminus used for purification. Top: image stained with Coomassie Brilliant Blue. Bottom: the same gel scanned for Cy5 fluorescence (prior to Coomassie staining). ( C ) In vitro transcription activity of RNAP. The concentrations of RNAP derivatives were as indicated. The DNA template (a PCR fragment spanning from −75 to +295 of the concensus T7 RNAP promoter), was used at 10 nM. RNA products were labeled by incorporation of α 32 P-ATP, resolved on a denaturing polyacrylamide gel, and imaged by autoradiography. The major bands at ∼295 nt are the expected run-off products. DOI: http://dx.doi.org/10.7554/eLife.01775.009

Techniques Used: Labeling, Activity Assay, SDS Page, Purification, Staining, Fluorescence, In Vitro, Polymerase Chain Reaction, Autoradiography

15) Product Images from "In Vitro Epsilon RNA-Dependent Protein Priming Activity of Human Hepatitis B Virus Polymerase"

Article Title: In Vitro Epsilon RNA-Dependent Protein Priming Activity of Human Hepatitis B Virus Polymerase

Journal: Journal of Virology

doi: 10.1128/JVI.07137-11

Analysis of DP protein priming products by Tdp2 cleavage of the phosphotyrosyl bond between DNA and protein. Purified DP bound to M2 antibody affinity beads was assayed for protein priming. Free nucleotides were then removed with extensive washing, and priming products were mock treated (−) or treated with Tdp2 (+) to cleave the phosphotyrosyl-DNA linkages between DP and the linked nucleotides or DNA oligomers. The supernatant, which contained the released nucleotides/DNA, was collected and resolved on a urea–20% polyacrylamide gel (B). The beads, which contained the primed DP, were processed for SDS-PAGE to visualize the labeled DP (A). Radiolabeled proteins and nucleotides/DNA were detected by autoradiography. Priming was done in the presence of TMgNK buffer and [α- 32 P]dGTP (A, lanes 1 and 2; B, lanes 5 and 6) or TMnNK buffer and [α- 32 P]dGTP plus the unlabeled dCTP, TTP, and dATP (A, lanes 3 and 4; B, lanes 7 and 8). (C) [α- 32 P]dGTP stock was mock (lane 4) or apyrase treated (lane 5). The DP priming product obtained in TMgNK buffer and [α- 32 P]dGTP was either mock treated (lane 2) or Tdp2 treated (lane 3), which released dGMP from the DP-dGMP phosphotyrosyl linkage. Samples were resolved on a urea–20% polyacrylamide gel. The positions of 32 P-labeled 10-nucleotide marker (Invitrogen) (B) and DNA oligomers (dTG, dTGA, and dTGAA in panels B and C) are indicated, as are the positions of dGTP and dGMP. (D) HPLC analysis of dGTP and dGMP. (Panel 1) UV ( A 260 ) detection showing retention times of unlabeled dGMP and dGTP. (Panel 2) Detection of 32 P radioactivity from mock-treated DP priming products (−Tdp2), showing the absence of dGMP and the presence of residual dGTP substrate input. (Panel 3) Detection of 32 P radioactivity from Tdp2-treated DP priming products (+Tdp2), showing the presence of dGMP released by Tdp2 from DP and again some residual dGTP substrate input. The positions of dGMP and dGTP are indicated.
Figure Legend Snippet: Analysis of DP protein priming products by Tdp2 cleavage of the phosphotyrosyl bond between DNA and protein. Purified DP bound to M2 antibody affinity beads was assayed for protein priming. Free nucleotides were then removed with extensive washing, and priming products were mock treated (−) or treated with Tdp2 (+) to cleave the phosphotyrosyl-DNA linkages between DP and the linked nucleotides or DNA oligomers. The supernatant, which contained the released nucleotides/DNA, was collected and resolved on a urea–20% polyacrylamide gel (B). The beads, which contained the primed DP, were processed for SDS-PAGE to visualize the labeled DP (A). Radiolabeled proteins and nucleotides/DNA were detected by autoradiography. Priming was done in the presence of TMgNK buffer and [α- 32 P]dGTP (A, lanes 1 and 2; B, lanes 5 and 6) or TMnNK buffer and [α- 32 P]dGTP plus the unlabeled dCTP, TTP, and dATP (A, lanes 3 and 4; B, lanes 7 and 8). (C) [α- 32 P]dGTP stock was mock (lane 4) or apyrase treated (lane 5). The DP priming product obtained in TMgNK buffer and [α- 32 P]dGTP was either mock treated (lane 2) or Tdp2 treated (lane 3), which released dGMP from the DP-dGMP phosphotyrosyl linkage. Samples were resolved on a urea–20% polyacrylamide gel. The positions of 32 P-labeled 10-nucleotide marker (Invitrogen) (B) and DNA oligomers (dTG, dTGA, and dTGAA in panels B and C) are indicated, as are the positions of dGTP and dGMP. (D) HPLC analysis of dGTP and dGMP. (Panel 1) UV ( A 260 ) detection showing retention times of unlabeled dGMP and dGTP. (Panel 2) Detection of 32 P radioactivity from mock-treated DP priming products (−Tdp2), showing the absence of dGMP and the presence of residual dGTP substrate input. (Panel 3) Detection of 32 P radioactivity from Tdp2-treated DP priming products (+Tdp2), showing the presence of dGMP released by Tdp2 from DP and again some residual dGTP substrate input. The positions of dGMP and dGTP are indicated.

Techniques Used: Purification, SDS Page, Labeling, Autoradiography, Marker, High Performance Liquid Chromatography, Radioactivity

Detection of in vitro protein priming by purified HP. Priming reactions were performed by incubating immunoaffinity-purified HP with TMgNK buffer and [α- 32 P]dGTP (A to C ) or another labeled nucleotide as indicated (D and E). After priming, the beads were washed, and the labeled HP was resolved on an SDS–12.5% polyacrylamide gel. A priming reaction was also performed with the DHBV MiniRT2 (DP) in TMnNK buffer and resolved on the same gel for comparison (A, lane 1). Labeled HP and DP priming products were detected by autoradiography after SDS-PAGE. (A) In vitro priming reactions with WT (lanes 3 and 4) or mutant (lanes 5 and 6) HP with (lanes 4 to 6) or without Hε (lane 3) coexpression in cells. GFP + Hε (lane 2) represents priming using the control purification product from cells cotransfected with GFP and the Hε-expressing plasmid. (B) After protein priming, primed HP was untreated (−; lane 1) or treated with DNase I (D; lane 2) or pronase (P; lane 3) before analysis by SDS-PAGE. (C) The purified HP was mock treated (lane 1) or RNase treated (lane 2) before being used in protein priming. Labeled HP was detected by autoradiography after SDS-PAGE (top), and HP protein levels were measured by Western blotting using the anti-FLAG (α-Flag) antibody (bottom). (D) HP purified either with (lanes 5 to 8) or without (lanes 1 to 4) the coexpressed Hε was assayed for priming activity in the presence of [α- 32 P]dGTP (G; lanes 2 and 6), [α- 32 P]TTP (T; lanes 1 and 5), [α- 32 P]dCTP (C; lanes 3 and 7), or [α- 32 P]dATP (A; lanes 4 and 8). Priming signals were quantified via phosphorimaging, normalized to the highest signal (dGTP priming, set as 100%), and denoted below the lane numbers (as a percentage of dGTP signal). The labeled HP and DP priming products are indicated. (E) Shown on the top is a schematic diagram of the mutant Hε RNAs, with the last 4 nucleotides of the internal bulge and part of the upper stem, including its bottom A-U base pair. In Hε-B6G (left), the last (6th) bulge residue (i.e., B6) was changed (from rC in the WT) to rG and in Hε-B6A (right), the same residue was changed to rA. The mutated residues are highlighted in bold. Shown at the bottom are priming products obtained with the mutant Hε RNAs. The Hε-B6G (lanes 1 and 2) or -B6A (lanes 3 and 4) mutant was coexpressed with HP, and the purified HP-Hε complex was assayed for protein priming in vitro in the presence of the indicated 32 P-labeled nucleotide. The labeled HP priming products are indicated, as is the position of the protein molecular mass marker (in kDa).
Figure Legend Snippet: Detection of in vitro protein priming by purified HP. Priming reactions were performed by incubating immunoaffinity-purified HP with TMgNK buffer and [α- 32 P]dGTP (A to C ) or another labeled nucleotide as indicated (D and E). After priming, the beads were washed, and the labeled HP was resolved on an SDS–12.5% polyacrylamide gel. A priming reaction was also performed with the DHBV MiniRT2 (DP) in TMnNK buffer and resolved on the same gel for comparison (A, lane 1). Labeled HP and DP priming products were detected by autoradiography after SDS-PAGE. (A) In vitro priming reactions with WT (lanes 3 and 4) or mutant (lanes 5 and 6) HP with (lanes 4 to 6) or without Hε (lane 3) coexpression in cells. GFP + Hε (lane 2) represents priming using the control purification product from cells cotransfected with GFP and the Hε-expressing plasmid. (B) After protein priming, primed HP was untreated (−; lane 1) or treated with DNase I (D; lane 2) or pronase (P; lane 3) before analysis by SDS-PAGE. (C) The purified HP was mock treated (lane 1) or RNase treated (lane 2) before being used in protein priming. Labeled HP was detected by autoradiography after SDS-PAGE (top), and HP protein levels were measured by Western blotting using the anti-FLAG (α-Flag) antibody (bottom). (D) HP purified either with (lanes 5 to 8) or without (lanes 1 to 4) the coexpressed Hε was assayed for priming activity in the presence of [α- 32 P]dGTP (G; lanes 2 and 6), [α- 32 P]TTP (T; lanes 1 and 5), [α- 32 P]dCTP (C; lanes 3 and 7), or [α- 32 P]dATP (A; lanes 4 and 8). Priming signals were quantified via phosphorimaging, normalized to the highest signal (dGTP priming, set as 100%), and denoted below the lane numbers (as a percentage of dGTP signal). The labeled HP and DP priming products are indicated. (E) Shown on the top is a schematic diagram of the mutant Hε RNAs, with the last 4 nucleotides of the internal bulge and part of the upper stem, including its bottom A-U base pair. In Hε-B6G (left), the last (6th) bulge residue (i.e., B6) was changed (from rC in the WT) to rG and in Hε-B6A (right), the same residue was changed to rA. The mutated residues are highlighted in bold. Shown at the bottom are priming products obtained with the mutant Hε RNAs. The Hε-B6G (lanes 1 and 2) or -B6A (lanes 3 and 4) mutant was coexpressed with HP, and the purified HP-Hε complex was assayed for protein priming in vitro in the presence of the indicated 32 P-labeled nucleotide. The labeled HP priming products are indicated, as is the position of the protein molecular mass marker (in kDa).

Techniques Used: In Vitro, Purification, Labeling, Autoradiography, SDS Page, Mutagenesis, Expressing, Plasmid Preparation, Western Blot, Activity Assay, Marker

Differentiation of priming initiation from DNA polymerization by S1 nuclease digestion. (A) Protein priming was conducted with DP bound to M2 affinity beads in TMnNK buffer, in the presence of [α- 32 P]dGTP and unlabeled dCTP, dATP, and TTP. Priming products were either mock treated (−; lanes 5 and 6) or S1 treated (+; lanes 7 and 8), followed by mock treatment (−; lanes 5 and 7) or Tdp2 treatment (+; lanes 6 and 8), as described in Materials and Methods. Released nucleotides or DNAs were resolved by urea-PAGE and detected by autoradiography. The 10-nucleotide marker, the dTG, dTGA, and dTGAA DNA oligomers, and dGMP positions are indicated, as is the priming initiation product (I; i.e., the single dGMP residue released by Tdp2 from DP) or polymerization products (P; DNA polymerization from the first dGMP residue). (B) Protein priming was performed with DP in TMnNK buffer with [α- 32 P]dGTP (lanes 1 and 2) or with unlabeled dGTP (unlabled dNTP denoted by parentheses) followed by the addition of [α- 32 P]TTP to extend the unlabeled DP-dGMP initiation product (lanes 3 and 4). The priming products were then mock treated (−; lanes 1 and 3) or treated with S1 nuclease (+; lanes 2 and 4), resolved by SDS-PAGE, and detected by autoradiography. (C) Priming was performed with DP (lanes 1 and 2) or HP (lanes 3 to 6) in TMgNK buffer with [α- 32 P]dGTP (lanes 1 to 4) or with unlabeled dGTP first followed by addition of [α- 32 P]dATP to extend the unlabeled HP-dGMP initiation product (lanes 5 and 6). The priming products were either mock treated (−; lanes 1, 3, and 5) or S1 treated (+; lanes 2, 4, and 6), resolved by SDS-PAGE, and detected by autoradiography. (D) The percent decreases in DP and HP priming signals as a result of S1 nuclease treatment are represented. Mock-treated DP initiation reaction in the presence of [α- 32 P]dGTP alone, with either TMnNK or TMgNK buffer, was set as 100%, and the other reaction conditions, as explained in panels B and C, were normalized to this. The decrease in priming signal due to proteolytic degradation (unrelated to S1 nuclease cleavage of internucleotide linkages) was subtracted from the calculations. (E) DP or HP was incubated with or without S1 nuclease as described above. Protease degradation was monitored by Western blotting using the M2 anti-Flag antibody. HC, antibody heavy chain. The symbol * in panels B, C, and E represents DP and HP degradation products caused by contaminating protease activity in S1. Note that only some proteolytic degradation products detected by the Western blot (E) appeared to match the 32 P-labeled degradation products (B and C) since the labeled products must have contained the priming site(s), whereas the Western blot detected only fragments containing the N-terminal FLAG tag. Also, some labeled degradation products might be present at such low levels that they were undetectable by Western blotting. Note also that the appearance of the proteolytic degradation products was accompanied by the decrease of the full-length HP or DP in panels B, C, and E. (F) The diagram depicts the cleavage of the internucleotide linkages, but not the HP-dGMP linkage, by S1.
Figure Legend Snippet: Differentiation of priming initiation from DNA polymerization by S1 nuclease digestion. (A) Protein priming was conducted with DP bound to M2 affinity beads in TMnNK buffer, in the presence of [α- 32 P]dGTP and unlabeled dCTP, dATP, and TTP. Priming products were either mock treated (−; lanes 5 and 6) or S1 treated (+; lanes 7 and 8), followed by mock treatment (−; lanes 5 and 7) or Tdp2 treatment (+; lanes 6 and 8), as described in Materials and Methods. Released nucleotides or DNAs were resolved by urea-PAGE and detected by autoradiography. The 10-nucleotide marker, the dTG, dTGA, and dTGAA DNA oligomers, and dGMP positions are indicated, as is the priming initiation product (I; i.e., the single dGMP residue released by Tdp2 from DP) or polymerization products (P; DNA polymerization from the first dGMP residue). (B) Protein priming was performed with DP in TMnNK buffer with [α- 32 P]dGTP (lanes 1 and 2) or with unlabeled dGTP (unlabled dNTP denoted by parentheses) followed by the addition of [α- 32 P]TTP to extend the unlabeled DP-dGMP initiation product (lanes 3 and 4). The priming products were then mock treated (−; lanes 1 and 3) or treated with S1 nuclease (+; lanes 2 and 4), resolved by SDS-PAGE, and detected by autoradiography. (C) Priming was performed with DP (lanes 1 and 2) or HP (lanes 3 to 6) in TMgNK buffer with [α- 32 P]dGTP (lanes 1 to 4) or with unlabeled dGTP first followed by addition of [α- 32 P]dATP to extend the unlabeled HP-dGMP initiation product (lanes 5 and 6). The priming products were either mock treated (−; lanes 1, 3, and 5) or S1 treated (+; lanes 2, 4, and 6), resolved by SDS-PAGE, and detected by autoradiography. (D) The percent decreases in DP and HP priming signals as a result of S1 nuclease treatment are represented. Mock-treated DP initiation reaction in the presence of [α- 32 P]dGTP alone, with either TMnNK or TMgNK buffer, was set as 100%, and the other reaction conditions, as explained in panels B and C, were normalized to this. The decrease in priming signal due to proteolytic degradation (unrelated to S1 nuclease cleavage of internucleotide linkages) was subtracted from the calculations. (E) DP or HP was incubated with or without S1 nuclease as described above. Protease degradation was monitored by Western blotting using the M2 anti-Flag antibody. HC, antibody heavy chain. The symbol * in panels B, C, and E represents DP and HP degradation products caused by contaminating protease activity in S1. Note that only some proteolytic degradation products detected by the Western blot (E) appeared to match the 32 P-labeled degradation products (B and C) since the labeled products must have contained the priming site(s), whereas the Western blot detected only fragments containing the N-terminal FLAG tag. Also, some labeled degradation products might be present at such low levels that they were undetectable by Western blotting. Note also that the appearance of the proteolytic degradation products was accompanied by the decrease of the full-length HP or DP in panels B, C, and E. (F) The diagram depicts the cleavage of the internucleotide linkages, but not the HP-dGMP linkage, by S1.

Techniques Used: Polyacrylamide Gel Electrophoresis, Autoradiography, Marker, SDS Page, Incubation, Western Blot, Activity Assay, Labeling, FLAG-tag

Analysis of HP protein priming products by Tdp2 cleavage of the phosphotyrosyl bond between DNA and protein. Purified HP bound to M2 antibody affinity beads was assayed for protein priming. Free nucleotides were then removed with extensive washing, and priming products were mock treated (−) or treated with Tdp2 (+) to cleave the phosphotyrosyl-DNA linkages between HP and the linked nucleotides or DNA oligomers. The supernatant, which contained the released nucleotides/DNA, was collected and resolved on a urea–20% polyacrylamide gel (B to D). The beads, which contained the primed HP, were processed for SDS-PAGE to visualize the labeled HP (A). Radiolabeled proteins and nucleotides/DNA were detected by autoradiography. Priming was done in the presence of [α- 32 P]dGTP (A, lanes 1 and 2; B, lanes 3 and 4), [α- 32 P]dATP (A, lanes 3 and 4; B, lanes 5 and 6), [α- 32 P]dGTP plus [α- 32 P]dATP (A, lanes 5 and 6; B, lanes 1 and 2; D, lanes 1 and 2), [α- 32 P]dGTP plus [α- 32 P]dTTP (D, lanes 3 and 4), [α- 32 P]dGTP plus unlabeled dATP (C, lanes 3 and 4), or the other three unlabeled dNTPs (C, lanes 5 and 6; denoted as N). Unlabeled dNTPs are denoted with parentheses in panel C. The positions of the 32 P-labeled 10-nucleotide marker (Invitrogen) (C) and DNA oligomers (dGA, dGAA, and dGAAA in panels B to D and dTG, dTGA, and dTGAA in panel C) are indicated, as are the positions of dGTP and dGMP. (E) The top diagram depicts the HP priming product, i.e., the dGAA DNA oligomer that is covalently attached to HP via Y63 and templated by the last three nucleotides (rUUC) of the internal bulge of Hε. Part of the upper stem of Hε, with its bottom A-U base pair, is also shown. The phosphotyrosyl protein-DNA linkage is specifically cleaved by Tdp2 as shown. The bottom diagram depicts DNA strand elongation following primer transfer, whereby the HP-dGAA complex is translocated from Hε to DR1, and the dGAA oligomer is further extended, potentially up to dGAAAAA in the presence of only dGTP and dATP. The putative dGAAAA or dGAAAAA product released by Tdp2 from HP is also denoted by “GAAAA(?)” in panel D.
Figure Legend Snippet: Analysis of HP protein priming products by Tdp2 cleavage of the phosphotyrosyl bond between DNA and protein. Purified HP bound to M2 antibody affinity beads was assayed for protein priming. Free nucleotides were then removed with extensive washing, and priming products were mock treated (−) or treated with Tdp2 (+) to cleave the phosphotyrosyl-DNA linkages between HP and the linked nucleotides or DNA oligomers. The supernatant, which contained the released nucleotides/DNA, was collected and resolved on a urea–20% polyacrylamide gel (B to D). The beads, which contained the primed HP, were processed for SDS-PAGE to visualize the labeled HP (A). Radiolabeled proteins and nucleotides/DNA were detected by autoradiography. Priming was done in the presence of [α- 32 P]dGTP (A, lanes 1 and 2; B, lanes 3 and 4), [α- 32 P]dATP (A, lanes 3 and 4; B, lanes 5 and 6), [α- 32 P]dGTP plus [α- 32 P]dATP (A, lanes 5 and 6; B, lanes 1 and 2; D, lanes 1 and 2), [α- 32 P]dGTP plus [α- 32 P]dTTP (D, lanes 3 and 4), [α- 32 P]dGTP plus unlabeled dATP (C, lanes 3 and 4), or the other three unlabeled dNTPs (C, lanes 5 and 6; denoted as N). Unlabeled dNTPs are denoted with parentheses in panel C. The positions of the 32 P-labeled 10-nucleotide marker (Invitrogen) (C) and DNA oligomers (dGA, dGAA, and dGAAA in panels B to D and dTG, dTGA, and dTGAA in panel C) are indicated, as are the positions of dGTP and dGMP. (E) The top diagram depicts the HP priming product, i.e., the dGAA DNA oligomer that is covalently attached to HP via Y63 and templated by the last three nucleotides (rUUC) of the internal bulge of Hε. Part of the upper stem of Hε, with its bottom A-U base pair, is also shown. The phosphotyrosyl protein-DNA linkage is specifically cleaved by Tdp2 as shown. The bottom diagram depicts DNA strand elongation following primer transfer, whereby the HP-dGAA complex is translocated from Hε to DR1, and the dGAA oligomer is further extended, potentially up to dGAAAAA in the presence of only dGTP and dATP. The putative dGAAAA or dGAAAAA product released by Tdp2 from HP is also denoted by “GAAAA(?)” in panel D.

Techniques Used: Purification, SDS Page, Labeling, Autoradiography, Marker

16) Product Images from "In Vitro Epsilon RNA-Dependent Protein Priming Activity of Human Hepatitis B Virus Polymerase"

Article Title: In Vitro Epsilon RNA-Dependent Protein Priming Activity of Human Hepatitis B Virus Polymerase

Journal: Journal of Virology

doi: 10.1128/JVI.07137-11

Analysis of DP protein priming products by Tdp2 cleavage of the phosphotyrosyl bond between DNA and protein. Purified DP bound to M2 antibody affinity beads was assayed for protein priming. Free nucleotides were then removed with extensive washing, and priming products were mock treated (−) or treated with Tdp2 (+) to cleave the phosphotyrosyl-DNA linkages between DP and the linked nucleotides or DNA oligomers. The supernatant, which contained the released nucleotides/DNA, was collected and resolved on a urea–20% polyacrylamide gel (B). The beads, which contained the primed DP, were processed for SDS-PAGE to visualize the labeled DP (A). Radiolabeled proteins and nucleotides/DNA were detected by autoradiography. Priming was done in the presence of TMgNK buffer and [α- 32 P]dGTP (A, lanes 1 and 2; B, lanes 5 and 6) or TMnNK buffer and [α- 32 P]dGTP plus the unlabeled dCTP, TTP, and dATP (A, lanes 3 and 4; B, lanes 7 and 8). (C) [α- 32 P]dGTP stock was mock (lane 4) or apyrase treated (lane 5). The DP priming product obtained in TMgNK buffer and [α- 32 P]dGTP was either mock treated (lane 2) or Tdp2 treated (lane 3), which released dGMP from the DP-dGMP phosphotyrosyl linkage. Samples were resolved on a urea–20% polyacrylamide gel. The positions of 32 P-labeled 10-nucleotide marker (Invitrogen) (B) and DNA oligomers (dTG, dTGA, and dTGAA in panels B and C) are indicated, as are the positions of dGTP and dGMP. (D) HPLC analysis of dGTP and dGMP. (Panel 1) UV ( A 260 ) detection showing retention times of unlabeled dGMP and dGTP. (Panel 2) Detection of 32 P radioactivity from mock-treated DP priming products (−Tdp2), showing the absence of dGMP and the presence of residual dGTP substrate input. (Panel 3) Detection of 32 P radioactivity from Tdp2-treated DP priming products (+Tdp2), showing the presence of dGMP released by Tdp2 from DP and again some residual dGTP substrate input. The positions of dGMP and dGTP are indicated.
Figure Legend Snippet: Analysis of DP protein priming products by Tdp2 cleavage of the phosphotyrosyl bond between DNA and protein. Purified DP bound to M2 antibody affinity beads was assayed for protein priming. Free nucleotides were then removed with extensive washing, and priming products were mock treated (−) or treated with Tdp2 (+) to cleave the phosphotyrosyl-DNA linkages between DP and the linked nucleotides or DNA oligomers. The supernatant, which contained the released nucleotides/DNA, was collected and resolved on a urea–20% polyacrylamide gel (B). The beads, which contained the primed DP, were processed for SDS-PAGE to visualize the labeled DP (A). Radiolabeled proteins and nucleotides/DNA were detected by autoradiography. Priming was done in the presence of TMgNK buffer and [α- 32 P]dGTP (A, lanes 1 and 2; B, lanes 5 and 6) or TMnNK buffer and [α- 32 P]dGTP plus the unlabeled dCTP, TTP, and dATP (A, lanes 3 and 4; B, lanes 7 and 8). (C) [α- 32 P]dGTP stock was mock (lane 4) or apyrase treated (lane 5). The DP priming product obtained in TMgNK buffer and [α- 32 P]dGTP was either mock treated (lane 2) or Tdp2 treated (lane 3), which released dGMP from the DP-dGMP phosphotyrosyl linkage. Samples were resolved on a urea–20% polyacrylamide gel. The positions of 32 P-labeled 10-nucleotide marker (Invitrogen) (B) and DNA oligomers (dTG, dTGA, and dTGAA in panels B and C) are indicated, as are the positions of dGTP and dGMP. (D) HPLC analysis of dGTP and dGMP. (Panel 1) UV ( A 260 ) detection showing retention times of unlabeled dGMP and dGTP. (Panel 2) Detection of 32 P radioactivity from mock-treated DP priming products (−Tdp2), showing the absence of dGMP and the presence of residual dGTP substrate input. (Panel 3) Detection of 32 P radioactivity from Tdp2-treated DP priming products (+Tdp2), showing the presence of dGMP released by Tdp2 from DP and again some residual dGTP substrate input. The positions of dGMP and dGTP are indicated.

Techniques Used: Purification, SDS Page, Labeling, Autoradiography, Marker, High Performance Liquid Chromatography, Radioactivity

Detection of in vitro protein priming by purified HP. Priming reactions were performed by incubating immunoaffinity-purified HP with TMgNK buffer and [α- 32 P]dGTP (A to C ) or another labeled nucleotide as indicated (D and E). After priming, the beads were washed, and the labeled HP was resolved on an SDS–12.5% polyacrylamide gel. A priming reaction was also performed with the DHBV MiniRT2 (DP) in TMnNK buffer and resolved on the same gel for comparison (A, lane 1). Labeled HP and DP priming products were detected by autoradiography after SDS-PAGE. (A) In vitro priming reactions with WT (lanes 3 and 4) or mutant (lanes 5 and 6) HP with (lanes 4 to 6) or without Hε (lane 3) coexpression in cells. GFP + Hε (lane 2) represents priming using the control purification product from cells cotransfected with GFP and the Hε-expressing plasmid. (B) After protein priming, primed HP was untreated (−; lane 1) or treated with DNase I (D; lane 2) or pronase (P; lane 3) before analysis by SDS-PAGE. (C) The purified HP was mock treated (lane 1) or RNase treated (lane 2) before being used in protein priming. Labeled HP was detected by autoradiography after SDS-PAGE (top), and HP protein levels were measured by Western blotting using the anti-FLAG (α-Flag) antibody (bottom). (D) HP purified either with (lanes 5 to 8) or without (lanes 1 to 4) the coexpressed Hε was assayed for priming activity in the presence of [α- 32 P]dGTP (G; lanes 2 and 6), [α- 32 P]TTP (T; lanes 1 and 5), [α- 32 P]dCTP (C; lanes 3 and 7), or [α- 32 P]dATP (A; lanes 4 and 8). Priming signals were quantified via phosphorimaging, normalized to the highest signal (dGTP priming, set as 100%), and denoted below the lane numbers (as a percentage of dGTP signal). The labeled HP and DP priming products are indicated. (E) Shown on the top is a schematic diagram of the mutant Hε RNAs, with the last 4 nucleotides of the internal bulge and part of the upper stem, including its bottom A-U base pair. In Hε-B6G (left), the last (6th) bulge residue (i.e., B6) was changed (from rC in the WT) to rG and in Hε-B6A (right), the same residue was changed to rA. The mutated residues are highlighted in bold. Shown at the bottom are priming products obtained with the mutant Hε RNAs. The Hε-B6G (lanes 1 and 2) or -B6A (lanes 3 and 4) mutant was coexpressed with HP, and the purified HP-Hε complex was assayed for protein priming in vitro in the presence of the indicated 32 P-labeled nucleotide. The labeled HP priming products are indicated, as is the position of the protein molecular mass marker (in kDa).
Figure Legend Snippet: Detection of in vitro protein priming by purified HP. Priming reactions were performed by incubating immunoaffinity-purified HP with TMgNK buffer and [α- 32 P]dGTP (A to C ) or another labeled nucleotide as indicated (D and E). After priming, the beads were washed, and the labeled HP was resolved on an SDS–12.5% polyacrylamide gel. A priming reaction was also performed with the DHBV MiniRT2 (DP) in TMnNK buffer and resolved on the same gel for comparison (A, lane 1). Labeled HP and DP priming products were detected by autoradiography after SDS-PAGE. (A) In vitro priming reactions with WT (lanes 3 and 4) or mutant (lanes 5 and 6) HP with (lanes 4 to 6) or without Hε (lane 3) coexpression in cells. GFP + Hε (lane 2) represents priming using the control purification product from cells cotransfected with GFP and the Hε-expressing plasmid. (B) After protein priming, primed HP was untreated (−; lane 1) or treated with DNase I (D; lane 2) or pronase (P; lane 3) before analysis by SDS-PAGE. (C) The purified HP was mock treated (lane 1) or RNase treated (lane 2) before being used in protein priming. Labeled HP was detected by autoradiography after SDS-PAGE (top), and HP protein levels were measured by Western blotting using the anti-FLAG (α-Flag) antibody (bottom). (D) HP purified either with (lanes 5 to 8) or without (lanes 1 to 4) the coexpressed Hε was assayed for priming activity in the presence of [α- 32 P]dGTP (G; lanes 2 and 6), [α- 32 P]TTP (T; lanes 1 and 5), [α- 32 P]dCTP (C; lanes 3 and 7), or [α- 32 P]dATP (A; lanes 4 and 8). Priming signals were quantified via phosphorimaging, normalized to the highest signal (dGTP priming, set as 100%), and denoted below the lane numbers (as a percentage of dGTP signal). The labeled HP and DP priming products are indicated. (E) Shown on the top is a schematic diagram of the mutant Hε RNAs, with the last 4 nucleotides of the internal bulge and part of the upper stem, including its bottom A-U base pair. In Hε-B6G (left), the last (6th) bulge residue (i.e., B6) was changed (from rC in the WT) to rG and in Hε-B6A (right), the same residue was changed to rA. The mutated residues are highlighted in bold. Shown at the bottom are priming products obtained with the mutant Hε RNAs. The Hε-B6G (lanes 1 and 2) or -B6A (lanes 3 and 4) mutant was coexpressed with HP, and the purified HP-Hε complex was assayed for protein priming in vitro in the presence of the indicated 32 P-labeled nucleotide. The labeled HP priming products are indicated, as is the position of the protein molecular mass marker (in kDa).

Techniques Used: In Vitro, Purification, Labeling, Autoradiography, SDS Page, Mutagenesis, Expressing, Plasmid Preparation, Western Blot, Activity Assay, Marker

Differentiation of priming initiation from DNA polymerization by S1 nuclease digestion. (A) Protein priming was conducted with DP bound to M2 affinity beads in TMnNK buffer, in the presence of [α- 32 P]dGTP and unlabeled dCTP, dATP, and TTP. Priming products were either mock treated (−; lanes 5 and 6) or S1 treated (+; lanes 7 and 8), followed by mock treatment (−; lanes 5 and 7) or Tdp2 treatment (+; lanes 6 and 8), as described in Materials and Methods. Released nucleotides or DNAs were resolved by urea-PAGE and detected by autoradiography. The 10-nucleotide marker, the dTG, dTGA, and dTGAA DNA oligomers, and dGMP positions are indicated, as is the priming initiation product (I; i.e., the single dGMP residue released by Tdp2 from DP) or polymerization products (P; DNA polymerization from the first dGMP residue). (B) Protein priming was performed with DP in TMnNK buffer with [α- 32 P]dGTP (lanes 1 and 2) or with unlabeled dGTP (unlabled dNTP denoted by parentheses) followed by the addition of [α- 32 P]TTP to extend the unlabeled DP-dGMP initiation product (lanes 3 and 4). The priming products were then mock treated (−; lanes 1 and 3) or treated with S1 nuclease (+; lanes 2 and 4), resolved by SDS-PAGE, and detected by autoradiography. (C) Priming was performed with DP (lanes 1 and 2) or HP (lanes 3 to 6) in TMgNK buffer with [α- 32 P]dGTP (lanes 1 to 4) or with unlabeled dGTP first followed by addition of [α- 32 P]dATP to extend the unlabeled HP-dGMP initiation product (lanes 5 and 6). The priming products were either mock treated (−; lanes 1, 3, and 5) or S1 treated (+; lanes 2, 4, and 6), resolved by SDS-PAGE, and detected by autoradiography. (D) The percent decreases in DP and HP priming signals as a result of S1 nuclease treatment are represented. Mock-treated DP initiation reaction in the presence of [α- 32 P]dGTP alone, with either TMnNK or TMgNK buffer, was set as 100%, and the other reaction conditions, as explained in panels B and C, were normalized to this. The decrease in priming signal due to proteolytic degradation (unrelated to S1 nuclease cleavage of internucleotide linkages) was subtracted from the calculations. (E) DP or HP was incubated with or without S1 nuclease as described above. Protease degradation was monitored by Western blotting using the M2 anti-Flag antibody. HC, antibody heavy chain. The symbol * in panels B, C, and E represents DP and HP degradation products caused by contaminating protease activity in S1. Note that only some proteolytic degradation products detected by the Western blot (E) appeared to match the 32 P-labeled degradation products (B and C) since the labeled products must have contained the priming site(s), whereas the Western blot detected only fragments containing the N-terminal FLAG tag. Also, some labeled degradation products might be present at such low levels that they were undetectable by Western blotting. Note also that the appearance of the proteolytic degradation products was accompanied by the decrease of the full-length HP or DP in panels B, C, and E. (F) The diagram depicts the cleavage of the internucleotide linkages, but not the HP-dGMP linkage, by S1.
Figure Legend Snippet: Differentiation of priming initiation from DNA polymerization by S1 nuclease digestion. (A) Protein priming was conducted with DP bound to M2 affinity beads in TMnNK buffer, in the presence of [α- 32 P]dGTP and unlabeled dCTP, dATP, and TTP. Priming products were either mock treated (−; lanes 5 and 6) or S1 treated (+; lanes 7 and 8), followed by mock treatment (−; lanes 5 and 7) or Tdp2 treatment (+; lanes 6 and 8), as described in Materials and Methods. Released nucleotides or DNAs were resolved by urea-PAGE and detected by autoradiography. The 10-nucleotide marker, the dTG, dTGA, and dTGAA DNA oligomers, and dGMP positions are indicated, as is the priming initiation product (I; i.e., the single dGMP residue released by Tdp2 from DP) or polymerization products (P; DNA polymerization from the first dGMP residue). (B) Protein priming was performed with DP in TMnNK buffer with [α- 32 P]dGTP (lanes 1 and 2) or with unlabeled dGTP (unlabled dNTP denoted by parentheses) followed by the addition of [α- 32 P]TTP to extend the unlabeled DP-dGMP initiation product (lanes 3 and 4). The priming products were then mock treated (−; lanes 1 and 3) or treated with S1 nuclease (+; lanes 2 and 4), resolved by SDS-PAGE, and detected by autoradiography. (C) Priming was performed with DP (lanes 1 and 2) or HP (lanes 3 to 6) in TMgNK buffer with [α- 32 P]dGTP (lanes 1 to 4) or with unlabeled dGTP first followed by addition of [α- 32 P]dATP to extend the unlabeled HP-dGMP initiation product (lanes 5 and 6). The priming products were either mock treated (−; lanes 1, 3, and 5) or S1 treated (+; lanes 2, 4, and 6), resolved by SDS-PAGE, and detected by autoradiography. (D) The percent decreases in DP and HP priming signals as a result of S1 nuclease treatment are represented. Mock-treated DP initiation reaction in the presence of [α- 32 P]dGTP alone, with either TMnNK or TMgNK buffer, was set as 100%, and the other reaction conditions, as explained in panels B and C, were normalized to this. The decrease in priming signal due to proteolytic degradation (unrelated to S1 nuclease cleavage of internucleotide linkages) was subtracted from the calculations. (E) DP or HP was incubated with or without S1 nuclease as described above. Protease degradation was monitored by Western blotting using the M2 anti-Flag antibody. HC, antibody heavy chain. The symbol * in panels B, C, and E represents DP and HP degradation products caused by contaminating protease activity in S1. Note that only some proteolytic degradation products detected by the Western blot (E) appeared to match the 32 P-labeled degradation products (B and C) since the labeled products must have contained the priming site(s), whereas the Western blot detected only fragments containing the N-terminal FLAG tag. Also, some labeled degradation products might be present at such low levels that they were undetectable by Western blotting. Note also that the appearance of the proteolytic degradation products was accompanied by the decrease of the full-length HP or DP in panels B, C, and E. (F) The diagram depicts the cleavage of the internucleotide linkages, but not the HP-dGMP linkage, by S1.

Techniques Used: Polyacrylamide Gel Electrophoresis, Autoradiography, Marker, SDS Page, Incubation, Western Blot, Activity Assay, Labeling, FLAG-tag

Analysis of HP protein priming products by Tdp2 cleavage of the phosphotyrosyl bond between DNA and protein. Purified HP bound to M2 antibody affinity beads was assayed for protein priming. Free nucleotides were then removed with extensive washing, and priming products were mock treated (−) or treated with Tdp2 (+) to cleave the phosphotyrosyl-DNA linkages between HP and the linked nucleotides or DNA oligomers. The supernatant, which contained the released nucleotides/DNA, was collected and resolved on a urea–20% polyacrylamide gel (B to D). The beads, which contained the primed HP, were processed for SDS-PAGE to visualize the labeled HP (A). Radiolabeled proteins and nucleotides/DNA were detected by autoradiography. Priming was done in the presence of [α- 32 P]dGTP (A, lanes 1 and 2; B, lanes 3 and 4), [α- 32 P]dATP (A, lanes 3 and 4; B, lanes 5 and 6), [α- 32 P]dGTP plus [α- 32 P]dATP (A, lanes 5 and 6; B, lanes 1 and 2; D, lanes 1 and 2), [α- 32 P]dGTP plus [α- 32 P]dTTP (D, lanes 3 and 4), [α- 32 P]dGTP plus unlabeled dATP (C, lanes 3 and 4), or the other three unlabeled dNTPs (C, lanes 5 and 6; denoted as N). Unlabeled dNTPs are denoted with parentheses in panel C. The positions of the 32 P-labeled 10-nucleotide marker (Invitrogen) (C) and DNA oligomers (dGA, dGAA, and dGAAA in panels B to D and dTG, dTGA, and dTGAA in panel C) are indicated, as are the positions of dGTP and dGMP. (E) The top diagram depicts the HP priming product, i.e., the dGAA DNA oligomer that is covalently attached to HP via Y63 and templated by the last three nucleotides (rUUC) of the internal bulge of Hε. Part of the upper stem of Hε, with its bottom A-U base pair, is also shown. The phosphotyrosyl protein-DNA linkage is specifically cleaved by Tdp2 as shown. The bottom diagram depicts DNA strand elongation following primer transfer, whereby the HP-dGAA complex is translocated from Hε to DR1, and the dGAA oligomer is further extended, potentially up to dGAAAAA in the presence of only dGTP and dATP. The putative dGAAAA or dGAAAAA product released by Tdp2 from HP is also denoted by “GAAAA(?)” in panel D.
Figure Legend Snippet: Analysis of HP protein priming products by Tdp2 cleavage of the phosphotyrosyl bond between DNA and protein. Purified HP bound to M2 antibody affinity beads was assayed for protein priming. Free nucleotides were then removed with extensive washing, and priming products were mock treated (−) or treated with Tdp2 (+) to cleave the phosphotyrosyl-DNA linkages between HP and the linked nucleotides or DNA oligomers. The supernatant, which contained the released nucleotides/DNA, was collected and resolved on a urea–20% polyacrylamide gel (B to D). The beads, which contained the primed HP, were processed for SDS-PAGE to visualize the labeled HP (A). Radiolabeled proteins and nucleotides/DNA were detected by autoradiography. Priming was done in the presence of [α- 32 P]dGTP (A, lanes 1 and 2; B, lanes 3 and 4), [α- 32 P]dATP (A, lanes 3 and 4; B, lanes 5 and 6), [α- 32 P]dGTP plus [α- 32 P]dATP (A, lanes 5 and 6; B, lanes 1 and 2; D, lanes 1 and 2), [α- 32 P]dGTP plus [α- 32 P]dTTP (D, lanes 3 and 4), [α- 32 P]dGTP plus unlabeled dATP (C, lanes 3 and 4), or the other three unlabeled dNTPs (C, lanes 5 and 6; denoted as N). Unlabeled dNTPs are denoted with parentheses in panel C. The positions of the 32 P-labeled 10-nucleotide marker (Invitrogen) (C) and DNA oligomers (dGA, dGAA, and dGAAA in panels B to D and dTG, dTGA, and dTGAA in panel C) are indicated, as are the positions of dGTP and dGMP. (E) The top diagram depicts the HP priming product, i.e., the dGAA DNA oligomer that is covalently attached to HP via Y63 and templated by the last three nucleotides (rUUC) of the internal bulge of Hε. Part of the upper stem of Hε, with its bottom A-U base pair, is also shown. The phosphotyrosyl protein-DNA linkage is specifically cleaved by Tdp2 as shown. The bottom diagram depicts DNA strand elongation following primer transfer, whereby the HP-dGAA complex is translocated from Hε to DR1, and the dGAA oligomer is further extended, potentially up to dGAAAAA in the presence of only dGTP and dATP. The putative dGAAAA or dGAAAAA product released by Tdp2 from HP is also denoted by “GAAAA(?)” in panel D.

Techniques Used: Purification, SDS Page, Labeling, Autoradiography, Marker

17) Product Images from "A cancer-associated point mutation disables the steric gate of human PrimPol"

Article Title: A cancer-associated point mutation disables the steric gate of human PrimPol

Journal: Scientific Reports

doi: 10.1038/s41598-018-37439-0

Ribonucleotides are valid substrates for the Y100H variant during primer synthesis. ( a ) Scheme on the top shows PrimPol in complex with the GTCA template oligonucleotide and the two nucleotides forming the initial dimer. The autoradiograph shows dimer formation (primase activity) either by wild-type (WT) PrimPol or Y100H (400 nM) using [α- 32 P]dATP (upper panel) or [γ- 32 P] ATP (lower panel) as the 5′-site nucleotide (16 nM), and increasing concentrations of either dGTP or GTP as the incoming 3′-site nucleotide (0, 10, 50, 100 µM). ( b ) Binary complex formation, measured by EMSA, between WT PrimPol or Y100H and labeled 60-mer DNA template GTCC (1 nM), using the indicated PrimPol concentration (2.5, 5, 10, 20, 40 and 80 nM) ( c ) Pre-ternary complex formation measured by EMSA between WT PrimPol or Y100H (1 µM), 60-mer DNA template GTCC and either [α- 32 P]dGTP or [α- 32 P] GTP (16 nM). ( d ) DNA or RNA primers synthesized using as template 5′-T 20 ACGACAGACTGT 29 -3′ to allow elongation beyond the dimer. Products were labeled with [γ- 32 P] ATP . The autoradiographs shown in this figure are representative of at least 3 independent experiments.
Figure Legend Snippet: Ribonucleotides are valid substrates for the Y100H variant during primer synthesis. ( a ) Scheme on the top shows PrimPol in complex with the GTCA template oligonucleotide and the two nucleotides forming the initial dimer. The autoradiograph shows dimer formation (primase activity) either by wild-type (WT) PrimPol or Y100H (400 nM) using [α- 32 P]dATP (upper panel) or [γ- 32 P] ATP (lower panel) as the 5′-site nucleotide (16 nM), and increasing concentrations of either dGTP or GTP as the incoming 3′-site nucleotide (0, 10, 50, 100 µM). ( b ) Binary complex formation, measured by EMSA, between WT PrimPol or Y100H and labeled 60-mer DNA template GTCC (1 nM), using the indicated PrimPol concentration (2.5, 5, 10, 20, 40 and 80 nM) ( c ) Pre-ternary complex formation measured by EMSA between WT PrimPol or Y100H (1 µM), 60-mer DNA template GTCC and either [α- 32 P]dGTP or [α- 32 P] GTP (16 nM). ( d ) DNA or RNA primers synthesized using as template 5′-T 20 ACGACAGACTGT 29 -3′ to allow elongation beyond the dimer. Products were labeled with [γ- 32 P] ATP . The autoradiographs shown in this figure are representative of at least 3 independent experiments.

Techniques Used: Variant Assay, Autoradiography, Activity Assay, Labeling, Concentration Assay, Synthesized

18) Product Images from "A semisynthetic organism engineered for the stable expansion of the genetic alphabet"

Article Title: A semisynthetic organism engineered for the stable expansion of the genetic alphabet

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

doi: 10.1073/pnas.1616443114

The dATP uptake and growth of cells expressing Pt NTT2 as a function of inducer (IPTG) concentration or promoter strength, strain background, and presence of N-terminal signal sequences. ( A ) Uptake of [α- 32 P]-dATP in strains with inducible Pt NTT2.
Figure Legend Snippet: The dATP uptake and growth of cells expressing Pt NTT2 as a function of inducer (IPTG) concentration or promoter strength, strain background, and presence of N-terminal signal sequences. ( A ) Uptake of [α- 32 P]-dATP in strains with inducible Pt NTT2.

Techniques Used: Expressing, Concentration Assay

UBPs and transporter optimization. ( A ) Chemical structure of the d NaM -d 5SICS and d NaM -d TPT3 UBPs with a natural dC–dG base pair included for comparison. ( B ) Comparison of fitness and [α- 32 P]-dATP uptake in DM1 and the various constructed
Figure Legend Snippet: UBPs and transporter optimization. ( A ) Chemical structure of the d NaM -d 5SICS and d NaM -d TPT3 UBPs with a natural dC–dG base pair included for comparison. ( B ) Comparison of fitness and [α- 32 P]-dATP uptake in DM1 and the various constructed

Techniques Used: Construct

19) Product Images from "Bacillus subtilis RarA modulates replication restart"

Article Title: Bacillus subtilis RarA modulates replication restart

Journal: Nucleic Acids Research

doi: 10.1093/nar/gky541

RarA has no effect on ongoing DNA replication. ( A ) Scheme of the experimental design. The B. subtilis replisome was assembled on the DNA in the absence of RarA and in the presence of limiting ATPγS and then DNA replication was started by dNTP (including [α- 32 P]-dCTP) and ATP addition. After 20 s of initiating the reaction, 100 nM RarA was added or not, and reactions were continued for the indicated times. ( B ) Quantification of leading strand synthesis (mean ± SEM of > 3 independent experiments). ( C ) The leading strand DNA products obtained in one of these assays are visualized by denaturing gel electrophoresis and autoradiography.
Figure Legend Snippet: RarA has no effect on ongoing DNA replication. ( A ) Scheme of the experimental design. The B. subtilis replisome was assembled on the DNA in the absence of RarA and in the presence of limiting ATPγS and then DNA replication was started by dNTP (including [α- 32 P]-dCTP) and ATP addition. After 20 s of initiating the reaction, 100 nM RarA was added or not, and reactions were continued for the indicated times. ( B ) Quantification of leading strand synthesis (mean ± SEM of > 3 independent experiments). ( C ) The leading strand DNA products obtained in one of these assays are visualized by denaturing gel electrophoresis and autoradiography.

Techniques Used: Nucleic Acid Electrophoresis, Autoradiography

RarA does not inhibit SPP1 DNA replication. Quantification of leading ( A ) and lagging ( B ) strand synthesis obtained in standard SPP1 rolling circle DNA replication assays in the absence or in the presence of 100 nM RarA. Reaction mixes contained the SPP1 replisome, which is composed by SPP1 preprimosomal proteins (G 38 P and G 39 P) and DNA helicase G 40 P, and host proteins (DnaG, τ-complex, β, PolC and DnaE). The SPP1 replisome works with both SSB proteins (SsbA or G 36 P) and the effect of RarA on reactions having either viral G 36 P or host SbsA was tested. An enzyme mix consisting of all proteins except the SSB was generated, and added to a substrate mix composed of template DNA, rNTPs, dNTPs, and the indicated SSB (none, 30 nM G 36 P, or, 90 nM SsbA). Then reactions were placed at 37°C and incubated for 10 min. Leading strand synthesis was quantified by [α- 32 P]-dCTP incorporation and lagging strand synthesis by [α- 32 P]-dGTP incorporation. The results are expressed as the mean ± SEM of > 3 independent experiments.
Figure Legend Snippet: RarA does not inhibit SPP1 DNA replication. Quantification of leading ( A ) and lagging ( B ) strand synthesis obtained in standard SPP1 rolling circle DNA replication assays in the absence or in the presence of 100 nM RarA. Reaction mixes contained the SPP1 replisome, which is composed by SPP1 preprimosomal proteins (G 38 P and G 39 P) and DNA helicase G 40 P, and host proteins (DnaG, τ-complex, β, PolC and DnaE). The SPP1 replisome works with both SSB proteins (SsbA or G 36 P) and the effect of RarA on reactions having either viral G 36 P or host SbsA was tested. An enzyme mix consisting of all proteins except the SSB was generated, and added to a substrate mix composed of template DNA, rNTPs, dNTPs, and the indicated SSB (none, 30 nM G 36 P, or, 90 nM SsbA). Then reactions were placed at 37°C and incubated for 10 min. Leading strand synthesis was quantified by [α- 32 P]-dCTP incorporation and lagging strand synthesis by [α- 32 P]-dGTP incorporation. The results are expressed as the mean ± SEM of > 3 independent experiments.

Techniques Used: Generated, Incubation

SsbA-dependent RarA-mediated inhibition of B. subtilis PriA-dependent DNA replication. ( A ) Total DNA synthesis obtained in the presence of increasing RarA concentrations (15 min, 37°C). Reaction mixes contained all replisome components (preprimosomal proteins [PriA, DnaB, DnaD, DnaI), DnaC, DnaG, SsbA, τ-complex, β, PolC, DnaE), the indicated RarA concentration, template DNA, rNTPs, dNTPs and [α- 32 P]-dCTP and [α- 32 P]-dGTP. An enzyme mix consisting of all proteins except SsbA was generated and added to a substrate mix composed of template DNA, rNTPs, dNTPs, and SsbA. Then, samples were placed at 37°C. ( B ) Visualization of products obtained in the presence of 100 nM RarA or RarAK51A (15 min, 37°C). In the presence of [α- 32 P]-dCTP very large DNA fragments derived from rolling circle leading strand DNA synthesis is observed. A parallel reaction in the presence of [α- 32 P]-dGTP renders visible the small Okazaki fragments due to lagging strand DNA synthesis. Quantification of leading ( C ) and lagging strand ( D ) synthesis in the absence/presence of 100nM RarA and the indicated SsbA concentrations (15 min, 37°C). The quantification of the results is expressed as the mean ± SEM of six independent experiments. On the right part, a representative alkaline gel visualized by autoradiography showing the products of the DNA synthesis obtained in the presence or absence of RarA and SsbA.
Figure Legend Snippet: SsbA-dependent RarA-mediated inhibition of B. subtilis PriA-dependent DNA replication. ( A ) Total DNA synthesis obtained in the presence of increasing RarA concentrations (15 min, 37°C). Reaction mixes contained all replisome components (preprimosomal proteins [PriA, DnaB, DnaD, DnaI), DnaC, DnaG, SsbA, τ-complex, β, PolC, DnaE), the indicated RarA concentration, template DNA, rNTPs, dNTPs and [α- 32 P]-dCTP and [α- 32 P]-dGTP. An enzyme mix consisting of all proteins except SsbA was generated and added to a substrate mix composed of template DNA, rNTPs, dNTPs, and SsbA. Then, samples were placed at 37°C. ( B ) Visualization of products obtained in the presence of 100 nM RarA or RarAK51A (15 min, 37°C). In the presence of [α- 32 P]-dCTP very large DNA fragments derived from rolling circle leading strand DNA synthesis is observed. A parallel reaction in the presence of [α- 32 P]-dGTP renders visible the small Okazaki fragments due to lagging strand DNA synthesis. Quantification of leading ( C ) and lagging strand ( D ) synthesis in the absence/presence of 100nM RarA and the indicated SsbA concentrations (15 min, 37°C). The quantification of the results is expressed as the mean ± SEM of six independent experiments. On the right part, a representative alkaline gel visualized by autoradiography showing the products of the DNA synthesis obtained in the presence or absence of RarA and SsbA.

Techniques Used: Inhibition, DNA Synthesis, Concentration Assay, Generated, Derivative Assay, Autoradiography

20) Product Images from "Disruption of the developmentally-regulated Col2a1 pre-mRNA alternative splicing switch in a transgenic knock-in mouse model"

Article Title: Disruption of the developmentally-regulated Col2a1 pre-mRNA alternative splicing switch in a transgenic knock-in mouse model

Journal: Matrix Biology

doi: 10.1016/j.matbio.2011.12.004

Expression of Col2a1 mRNA alternatively-spliced isoforms in limb cartilage from knock-in mouse litters at different developmental time points. The autoradiograph shows [α- 32 P]dCTP- labeled cDNA bands representing endogenous Col2a1 isoforms in wild type (+/+), heterozygote (ki/+) and homozygote knock-in (ki/ki) hindlimb cartilage at different embryonic (E) and post-natal (P) time points of development (E12.5-P70). IIA and IIB cDNA band sizes are ~ 485bp and ~277bp, respectively. *Note that in WT and ki/+ cells, in addition to exon 2-containing IIA mRNA isoforms, levels of IID mRNA isoforms may also be present. Only IIA mRNA isoforms will be present in homozygous tissue.
Figure Legend Snippet: Expression of Col2a1 mRNA alternatively-spliced isoforms in limb cartilage from knock-in mouse litters at different developmental time points. The autoradiograph shows [α- 32 P]dCTP- labeled cDNA bands representing endogenous Col2a1 isoforms in wild type (+/+), heterozygote (ki/+) and homozygote knock-in (ki/ki) hindlimb cartilage at different embryonic (E) and post-natal (P) time points of development (E12.5-P70). IIA and IIB cDNA band sizes are ~ 485bp and ~277bp, respectively. *Note that in WT and ki/+ cells, in addition to exon 2-containing IIA mRNA isoforms, levels of IID mRNA isoforms may also be present. Only IIA mRNA isoforms will be present in homozygous tissue.

Techniques Used: Expressing, Knock-In, Autoradiography, Labeling

21) Product Images from "Independence between pre-mRNA splicing and DNA methylation in an isogenic minigene resource"

Article Title: Independence between pre-mRNA splicing and DNA methylation in an isogenic minigene resource

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkx900

Analysis of minigene induction and splicing. ( A ) Schematic of primer target locations for analysis of minigene-derived RNA. ( B ) Quantitative RT-PCR (qRT-PCR) with primers directed against Tnp1 exon 2 and the Pfn3 ORF following 4 h of treatment with Dox or vehicle. Minigene expression was normalized to the average of three reference genes ( ALDO, GAPDH and RPS16 ). Data shown are from three independent, biological replicates; error bars depict mean ± SEM. ( C ) Phosphorimaging analysis of 4 h induced RT-PCR products generated with intron-flanking Tnp1 primers or 5′ and 3′-directed Pfn3 ORF primers in the presence of dCTP α 32 P. Percent-spliced-in (PSI) was determined as the signal intensity of the spliced product versus the sum of spliced and unspliced products. Amplification of β-tubulin ( TUBB ) was performed in a multiplex reaction as an internal control for loading ( bottom ). ( D ) 3′-end RT-PCR to assess minigene polyadenylation. Distinct products reflect use of either the endogenous polyadenylation sites-encoded within the Tnp1 and Pfn3 minigenes or the vector-encoded site located within the BGH cassette. β-tubulin ( TUBB ) served as a loading control.
Figure Legend Snippet: Analysis of minigene induction and splicing. ( A ) Schematic of primer target locations for analysis of minigene-derived RNA. ( B ) Quantitative RT-PCR (qRT-PCR) with primers directed against Tnp1 exon 2 and the Pfn3 ORF following 4 h of treatment with Dox or vehicle. Minigene expression was normalized to the average of three reference genes ( ALDO, GAPDH and RPS16 ). Data shown are from three independent, biological replicates; error bars depict mean ± SEM. ( C ) Phosphorimaging analysis of 4 h induced RT-PCR products generated with intron-flanking Tnp1 primers or 5′ and 3′-directed Pfn3 ORF primers in the presence of dCTP α 32 P. Percent-spliced-in (PSI) was determined as the signal intensity of the spliced product versus the sum of spliced and unspliced products. Amplification of β-tubulin ( TUBB ) was performed in a multiplex reaction as an internal control for loading ( bottom ). ( D ) 3′-end RT-PCR to assess minigene polyadenylation. Distinct products reflect use of either the endogenous polyadenylation sites-encoded within the Tnp1 and Pfn3 minigenes or the vector-encoded site located within the BGH cassette. β-tubulin ( TUBB ) served as a loading control.

Techniques Used: Derivative Assay, Quantitative RT-PCR, Expressing, Reverse Transcription Polymerase Chain Reaction, Generated, Amplification, Multiplex Assay, Plasmid Preparation

22) Product Images from "A Novel Retinoic Acid-Responsive Element Regulates Retinoic Acid-Induced BLR1 Expression"

Article Title: A Novel Retinoic Acid-Responsive Element Regulates Retinoic Acid-Induced BLR1 Expression

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.24.6.2423-2443.2004

RA-induced nuclear extracts protect sequences in the distal region of the BLR1 promoter. A dsDNA fragment of 250 bp (spanning 217 bp [−1096 to −879] in the BLR1 promoter plus 25 bp at the 5′ end from the plasmid backbone sequence in the pBLR1-Luc promoter-reporter construct and 8 nt from the incorporated Eco RI and Pst I site) was prepared by PCR. After digestion with Eco RI and Pst I, the amplified fragment was [α- 32 P]dATP and [α- 32 P]dTTP end labeled at the 3′ recessed end with the Klenow fragment of Escherichia coli DNA polymerase I and used in the DNase I footprinting assay with nuclear extracts from HL-60 cells that were either left untreated (RA − ) or treated (RA + ) with all- trans -RA for 48 h. A DNA sequencing ladder (10 bp) was end labeled (using T4 polynucleotide kinase) with [γ- 32 P]ATP, heat denatured, and corun with the DNase I-treated samples as a size marker. The nucleotide sequence of the DNase I-protected site was determined by alignment of the protected region with the sequencing ladder. An approximately 17-bp region (−1071 to −1055) with the indicated sequence was specifically protected from DNase I digestion in the nuclear extracts from RA-treated cells. No footprint was visible with nuclear extracts from untreated cells. An autoradiograph of the DNA footprint is shown. The sizes of the denatured DNA sequence markers that were corun with the samples are indicated with arrows on the left side of the right panel. The 5′ and 3′ ends of the DNA probe used in the footprinting assay are indicated by arrows pointing up and down. The nucleotide sequence of the DNA footprint is shown on the right. Numbers indicate the positions of start and end points of the protection region relative to +1, the transcriptional initiation site.
Figure Legend Snippet: RA-induced nuclear extracts protect sequences in the distal region of the BLR1 promoter. A dsDNA fragment of 250 bp (spanning 217 bp [−1096 to −879] in the BLR1 promoter plus 25 bp at the 5′ end from the plasmid backbone sequence in the pBLR1-Luc promoter-reporter construct and 8 nt from the incorporated Eco RI and Pst I site) was prepared by PCR. After digestion with Eco RI and Pst I, the amplified fragment was [α- 32 P]dATP and [α- 32 P]dTTP end labeled at the 3′ recessed end with the Klenow fragment of Escherichia coli DNA polymerase I and used in the DNase I footprinting assay with nuclear extracts from HL-60 cells that were either left untreated (RA − ) or treated (RA + ) with all- trans -RA for 48 h. A DNA sequencing ladder (10 bp) was end labeled (using T4 polynucleotide kinase) with [γ- 32 P]ATP, heat denatured, and corun with the DNase I-treated samples as a size marker. The nucleotide sequence of the DNase I-protected site was determined by alignment of the protected region with the sequencing ladder. An approximately 17-bp region (−1071 to −1055) with the indicated sequence was specifically protected from DNase I digestion in the nuclear extracts from RA-treated cells. No footprint was visible with nuclear extracts from untreated cells. An autoradiograph of the DNA footprint is shown. The sizes of the denatured DNA sequence markers that were corun with the samples are indicated with arrows on the left side of the right panel. The 5′ and 3′ ends of the DNA probe used in the footprinting assay are indicated by arrows pointing up and down. The nucleotide sequence of the DNA footprint is shown on the right. Numbers indicate the positions of start and end points of the protection region relative to +1, the transcriptional initiation site.

Techniques Used: Plasmid Preparation, Sequencing, Construct, Polymerase Chain Reaction, Amplification, Labeling, Footprinting, DNA Sequencing, Marker, Autoradiography

23) Product Images from "Inhibition of translation by cytotrienin A--a member of the ansamycin family"

Article Title: Inhibition of translation by cytotrienin A--a member of the ansamycin family

Journal: RNA

doi: 10.1261/rna.2307710

Cyt A does not affect ternary formation. ( A ) Cyt A does not inhibit GTP binding to eEF1A. Purified eEF1A (1 μg) was UV cross-linked to [α 32 P]GTP in the presence (lanes 4–7 ) or absence (lanes 1–3 ) of Phe-tRNA Phe and 50 μM
Figure Legend Snippet: Cyt A does not affect ternary formation. ( A ) Cyt A does not inhibit GTP binding to eEF1A. Purified eEF1A (1 μg) was UV cross-linked to [α 32 P]GTP in the presence (lanes 4–7 ) or absence (lanes 1–3 ) of Phe-tRNA Phe and 50 μM

Techniques Used: Binding Assay, Purification

24) Product Images from "Novel (p)ppGpp Binding and Metabolizing Proteins of Escherichia coli"

Article Title: Novel (p)ppGpp Binding and Metabolizing Proteins of Escherichia coli

Journal: mBio

doi: 10.1128/mBio.02188-17

Translational GTPases are conserved targets of (p)ppGpp. (A) Competition assay of RF3 and Der (20 μM) and LepA (10 μM) binding [α- 32 P]ppGpp (2 nM) in the presence of cold competitors (100 μM). (B and C) Binding curves and K d determination of RF3 (B) and Der (C) binding of α- 32 P-labeled ppGpp, pppGpp, GTP, or GDP (2 nM [each]). At least three replicates were performed. The apparent K d values corresponding to each protein-ligand interaction are shown. (D) Dissociation curves for Der (50 μM) and [α- 32 P]ppGpp (2 nM) in the presence of either ppGpp or GTP (100 μM) (cold). (E and F) Binding curves and K d determination for DerG1 (E) and SaDer (F) binding α- 32 P-labeled ppGpp, pppGpp, or GTP (2 nM [each]). At least three replicates were performed. The apparent K d values are shown for each protein-ligand interaction.
Figure Legend Snippet: Translational GTPases are conserved targets of (p)ppGpp. (A) Competition assay of RF3 and Der (20 μM) and LepA (10 μM) binding [α- 32 P]ppGpp (2 nM) in the presence of cold competitors (100 μM). (B and C) Binding curves and K d determination of RF3 (B) and Der (C) binding of α- 32 P-labeled ppGpp, pppGpp, GTP, or GDP (2 nM [each]). At least three replicates were performed. The apparent K d values corresponding to each protein-ligand interaction are shown. (D) Dissociation curves for Der (50 μM) and [α- 32 P]ppGpp (2 nM) in the presence of either ppGpp or GTP (100 μM) (cold). (E and F) Binding curves and K d determination for DerG1 (E) and SaDer (F) binding α- 32 P-labeled ppGpp, pppGpp, or GTP (2 nM [each]). At least three replicates were performed. The apparent K d values are shown for each protein-ligand interaction.

Techniques Used: Competitive Binding Assay, Binding Assay, Labeling

YgdH binds (p)ppGpp antagonistically with magnesium. (A) Competition assay of purified YgdH protein (20 μM) binding a 1:1 mixture of ppGpp and pppGpp (2 nM [each]) in the absence or presence of EDTA. Representative DRaCALA spots and quantifications (average values for bound fractions and standards errors of the means [SEM]) of binding signals are shown. (B) Thin-layer chromatography (TLC) of DRaCALA binding reactions determined by using 1.5 M K 2 HPO 4 (pH 3.4) as the mobile phase. Binding reactions performed with purified MutT, Der, or YgdH were run in parallel with standards of [α- 32 P]GTP and a mixture of [α- 32 P]ppGpp and [α- 32 P]pppGpp (2 nM [each]). (C) Binding curves and K d determinations for YgdH interacting with α- 32 P-labeled ppGpp, pppGpp, and GTP (2 nM [each]) without or with 1.5 mM MgCl 2 . The apparent K d values corresponding to each protein-ligand interaction are shown. (D) Magnesium (0 to 10.15 mM) IC 50 determinations of binding of [α- 32 P]ppGpp (2 nM) to YgdH (50 μM). IC 50 values are shown. (E) Competition assay of YgdH (50 μM) binding [α- 32 P]ppGpp (2 nM) in the presence of 100 μM cold competitors [including (p)ppGpp and the substrates of YgdH (GMP, AMP, and IMP)] without or with 1.5 mM magnesium.
Figure Legend Snippet: YgdH binds (p)ppGpp antagonistically with magnesium. (A) Competition assay of purified YgdH protein (20 μM) binding a 1:1 mixture of ppGpp and pppGpp (2 nM [each]) in the absence or presence of EDTA. Representative DRaCALA spots and quantifications (average values for bound fractions and standards errors of the means [SEM]) of binding signals are shown. (B) Thin-layer chromatography (TLC) of DRaCALA binding reactions determined by using 1.5 M K 2 HPO 4 (pH 3.4) as the mobile phase. Binding reactions performed with purified MutT, Der, or YgdH were run in parallel with standards of [α- 32 P]GTP and a mixture of [α- 32 P]ppGpp and [α- 32 P]pppGpp (2 nM [each]). (C) Binding curves and K d determinations for YgdH interacting with α- 32 P-labeled ppGpp, pppGpp, and GTP (2 nM [each]) without or with 1.5 mM MgCl 2 . The apparent K d values corresponding to each protein-ligand interaction are shown. (D) Magnesium (0 to 10.15 mM) IC 50 determinations of binding of [α- 32 P]ppGpp (2 nM) to YgdH (50 μM). IC 50 values are shown. (E) Competition assay of YgdH (50 μM) binding [α- 32 P]ppGpp (2 nM) in the presence of 100 μM cold competitors [including (p)ppGpp and the substrates of YgdH (GMP, AMP, and IMP)] without or with 1.5 mM magnesium.

Techniques Used: Competitive Binding Assay, Purification, Binding Assay, Thin Layer Chromatography, Labeling

In vitro cleavage of ppGpp by MutT, NudG, NadR, and TrmE. (A) Competition assay of whole-cell lysates containing overexpressed MutT and NudG binding [α- 32 P]ppGpp (2 nM) in the presence of cold competitors (100 μM). (B) Competition assay of purified NadR (left) and TrmE (right) (20 μM [each]) binding α- 32 P-labeled ppGpp and pppGpp (2 nM [each]) in the presence of cold competitors (100 μM). (C) DRaCALA spots of purified proteins (10 μM) binding a mixture of α- 32 P-labeled ppGpp and pppGpp (2 nM [each]) in the absence or presence of EDTA (25 mM). (D) TLC assessment of cleavage products from the binding reactions described for panel C. A mixture of ppGpp and pppGpp was run as the standard, and both molecules are indicated. (E) Quantification of (p)ppGpp percentage determined as described for panel D. (F) Competition assay of whole-cell lysates containing overproduced MutT and NudG binding [α- 32 P](p)ppGpp (2 nM) in the presence of cold competitors and their native substrates (100 μM [each]). Representative DRaCALA spots are shown. 8OdG, 8-oxo-dGTP; 8OG, 8-oxo-GTP; 2OdA, 2-hydroxyl-dATP; 2OA, 2-hydroxyl-ATP. (G) TLC assessment of cleavage products of [α- 32 ), and reactions were stopped by addition of excess EDTA (25 mM). pGp and ppGpp are indicated.
Figure Legend Snippet: In vitro cleavage of ppGpp by MutT, NudG, NadR, and TrmE. (A) Competition assay of whole-cell lysates containing overexpressed MutT and NudG binding [α- 32 P]ppGpp (2 nM) in the presence of cold competitors (100 μM). (B) Competition assay of purified NadR (left) and TrmE (right) (20 μM [each]) binding α- 32 P-labeled ppGpp and pppGpp (2 nM [each]) in the presence of cold competitors (100 μM). (C) DRaCALA spots of purified proteins (10 μM) binding a mixture of α- 32 P-labeled ppGpp and pppGpp (2 nM [each]) in the absence or presence of EDTA (25 mM). (D) TLC assessment of cleavage products from the binding reactions described for panel C. A mixture of ppGpp and pppGpp was run as the standard, and both molecules are indicated. (E) Quantification of (p)ppGpp percentage determined as described for panel D. (F) Competition assay of whole-cell lysates containing overproduced MutT and NudG binding [α- 32 P](p)ppGpp (2 nM) in the presence of cold competitors and their native substrates (100 μM [each]). Representative DRaCALA spots are shown. 8OdG, 8-oxo-dGTP; 8OG, 8-oxo-GTP; 2OdA, 2-hydroxyl-dATP; 2OA, 2-hydroxyl-ATP. (G) TLC assessment of cleavage products of [α- 32 ), and reactions were stopped by addition of excess EDTA (25 mM). pGp and ppGpp are indicated.

Techniques Used: In Vitro, Competitive Binding Assay, Binding Assay, Purification, Labeling, Thin Layer Chromatography

HypB specifically binds (p)ppGpp with physiological affinity. (A) Competition assay of HypB (20 μM) binding α- 32 P-labeled ppGpp and pppGpp (2 nM [each]) in the presence of cold competitors (100 μM). (B) Binding curves and K d determination for HypB binding α- 32 P-labeled ppGpp, pppGpp, and GTP (2 nM [each]). Three replicates were performed, and the apparent K d values are indicated.
Figure Legend Snippet: HypB specifically binds (p)ppGpp with physiological affinity. (A) Competition assay of HypB (20 μM) binding α- 32 P-labeled ppGpp and pppGpp (2 nM [each]) in the presence of cold competitors (100 μM). (B) Binding curves and K d determination for HypB binding α- 32 P-labeled ppGpp, pppGpp, and GTP (2 nM [each]). Three replicates were performed, and the apparent K d values are indicated.

Techniques Used: Competitive Binding Assay, Binding Assay, Labeling

GTP biosynthesis and salvage pathways are targeted by (p)ppGpp. (A) Schematic of purine biosynthesis pathways with (p)ppGpp targets highlighted by colored boxes. Green indicates E. coli targets identified here; blue indicates specific Bacillus / Staphylococcus targets; red indicates E. coli targets reported previously but not confirmed in this study; gray indicates a target found in E. coli , Bacillus , and Staphylococcus . G, guanine; X, xanthine; H, hypoxanthine; A, adenine; PRPP, phosphoribosyl pyrophosphate; Gln, glutamine. (B) Binding curves and apparent K d values for E. coli Gpt, Hpt, and Apt binding pppGpp and ppGpp (2 nM [each]). The average values for bound fractions and standard errors of the means (SEM) determined for at least three replicates were plotted and the curve-fitted and K d ). The apparent K d values corresponding to each protein-ligand interaction are shown. (C) Competition assay of Gpt, Hpt, and Apt (20 μM [each]) binding [α- 32 P]ppGpp (2 nM) in the presence of cold competitors (100 μM). The average values for bound fractions and standard errors of the means (SEM) determined for at least three replicates were plotted. Representative DRaCALA spots are shown above the respective diagrams.
Figure Legend Snippet: GTP biosynthesis and salvage pathways are targeted by (p)ppGpp. (A) Schematic of purine biosynthesis pathways with (p)ppGpp targets highlighted by colored boxes. Green indicates E. coli targets identified here; blue indicates specific Bacillus / Staphylococcus targets; red indicates E. coli targets reported previously but not confirmed in this study; gray indicates a target found in E. coli , Bacillus , and Staphylococcus . G, guanine; X, xanthine; H, hypoxanthine; A, adenine; PRPP, phosphoribosyl pyrophosphate; Gln, glutamine. (B) Binding curves and apparent K d values for E. coli Gpt, Hpt, and Apt binding pppGpp and ppGpp (2 nM [each]). The average values for bound fractions and standard errors of the means (SEM) determined for at least three replicates were plotted and the curve-fitted and K d ). The apparent K d values corresponding to each protein-ligand interaction are shown. (C) Competition assay of Gpt, Hpt, and Apt (20 μM [each]) binding [α- 32 P]ppGpp (2 nM) in the presence of cold competitors (100 μM). The average values for bound fractions and standard errors of the means (SEM) determined for at least three replicates were plotted. Representative DRaCALA spots are shown above the respective diagrams.

Techniques Used: Binding Assay, Competitive Binding Assay

25) Product Images from "Multisite phosphorylation of doublecortin by cyclin-dependent kinase 5"

Article Title: Multisite phosphorylation of doublecortin by cyclin-dependent kinase 5

Journal: Biochemical Journal

doi: 10.1042/BJ20040324

Distribution of the cdk5 phosphorylation sites in DCX ( a ) DCX was phosphorylated for 10 min in the presence of [γ- 32 P]ATP and for 90 min in the presence of excess non-radioactive ATP and the samples were mixed. After SDS/PAGE, DCX was digested with trypsin and the fragments were separated by HPLC. A plot of Cerenkov radiation versus fraction number for the reversed-phase HPLC separation is shown. The percentage concentration of the organic phase (phase B) is also shown on the right axis. The fractions that showed a specific increase in phosphorylation were pooled into seven (i–vii) samples. ( b – e ) Part of the MALDI-MS spectra of samples v and vi before (Control) and after their treatment with alkaline phosphatase to dephosphorylate the peptides. Sample v before ( b ) and after ( c ) dephosphorylation. An 80 Da mass shift from m / z 1507.70 to 1427.75 and m / z 1490.64 to 1410.64 indicates the loss of a phosphate group. The dephosphorylated peptides match the mass of DCX 331–344 and the N-terminal pyroglutamic acid modified DCX 331–344 respectively. A region of the MALDI-MS spectra from sample vi is shown before ( d ) and after ( e ) alkaline phosphatase treatment. The 80 Da mass shift from m / z 2072.82 to 1992.87 and m / z 2056.81 to 1976.86 indicates loss of a phosphate group. The dephosphorylated peptides match oxidized and non-oxidized DCX 23–39 respectively.
Figure Legend Snippet: Distribution of the cdk5 phosphorylation sites in DCX ( a ) DCX was phosphorylated for 10 min in the presence of [γ- 32 P]ATP and for 90 min in the presence of excess non-radioactive ATP and the samples were mixed. After SDS/PAGE, DCX was digested with trypsin and the fragments were separated by HPLC. A plot of Cerenkov radiation versus fraction number for the reversed-phase HPLC separation is shown. The percentage concentration of the organic phase (phase B) is also shown on the right axis. The fractions that showed a specific increase in phosphorylation were pooled into seven (i–vii) samples. ( b – e ) Part of the MALDI-MS spectra of samples v and vi before (Control) and after their treatment with alkaline phosphatase to dephosphorylate the peptides. Sample v before ( b ) and after ( c ) dephosphorylation. An 80 Da mass shift from m / z 1507.70 to 1427.75 and m / z 1490.64 to 1410.64 indicates the loss of a phosphate group. The dephosphorylated peptides match the mass of DCX 331–344 and the N-terminal pyroglutamic acid modified DCX 331–344 respectively. A region of the MALDI-MS spectra from sample vi is shown before ( d ) and after ( e ) alkaline phosphatase treatment. The 80 Da mass shift from m / z 2072.82 to 1992.87 and m / z 2056.81 to 1976.86 indicates loss of a phosphate group. The dephosphorylated peptides match oxidized and non-oxidized DCX 23–39 respectively.

Techniques Used: SDS Page, High Performance Liquid Chromatography, Concentration Assay, Mass Spectrometry, De-Phosphorylation Assay, Modification

Comparative phosphorylation of wt and mutant DCX by cdk5 GST-fusion protein (approx. 1 μg) of wt DCX, serine to alanine mutants and DCX truncations was phosphorylated by recombinant GST-cdk5/p25 in the presence of [γ- 32 or 33 P]ATP for 5 min at 37 °C and then analysed by SDS/PAGE and autoradiography. Phosphorylation was quantified by a STORM PhosphorImager. Results are represented as a percentage of wt DCX for the corrected activity (see the Methods section). Means±S.E.M. for six or seven replicated reactions per protein are presented.
Figure Legend Snippet: Comparative phosphorylation of wt and mutant DCX by cdk5 GST-fusion protein (approx. 1 μg) of wt DCX, serine to alanine mutants and DCX truncations was phosphorylated by recombinant GST-cdk5/p25 in the presence of [γ- 32 or 33 P]ATP for 5 min at 37 °C and then analysed by SDS/PAGE and autoradiography. Phosphorylation was quantified by a STORM PhosphorImager. Results are represented as a percentage of wt DCX for the corrected activity (see the Methods section). Means±S.E.M. for six or seven replicated reactions per protein are presented.

Techniques Used: Mutagenesis, Recombinant, SDS Page, Autoradiography, Activity Assay

26) Product Images from "A smart thermo- and pH-responsive microfiltration membrane based on three-dimensional inverse colloidal crystals"

Article Title: A smart thermo- and pH-responsive microfiltration membrane based on three-dimensional inverse colloidal crystals

Journal: Scientific Reports

doi: 10.1038/s41598-017-12426-z

Ion transportation properties of the ICC membrane with different NIPAM and MAA content at different temperatures and pH values measured through conductivity variation in the downstream.
Figure Legend Snippet: Ion transportation properties of the ICC membrane with different NIPAM and MAA content at different temperatures and pH values measured through conductivity variation in the downstream.

Techniques Used: Immunocytochemistry

IR spectra of the co-polymers P(NIPAM-MAA-GMA) contains 50% ( a ), 30% ( b ) and 0% ( c ) of NIPAM and MAA.
Figure Legend Snippet: IR spectra of the co-polymers P(NIPAM-MAA-GMA) contains 50% ( a ), 30% ( b ) and 0% ( c ) of NIPAM and MAA.

Techniques Used:

( a ) Schematically illustration of the preparation of thermo-and pH-responsive ICC macroporous column; ( b ) Chemical structures of MAA and NIPAM.
Figure Legend Snippet: ( a ) Schematically illustration of the preparation of thermo-and pH-responsive ICC macroporous column; ( b ) Chemical structures of MAA and NIPAM.

Techniques Used: Immunocytochemistry

27) Product Images from "Purification and characterisation of the yeast plasma membrane ATP binding cassette transporter Pdr11p"

Article Title: Purification and characterisation of the yeast plasma membrane ATP binding cassette transporter Pdr11p

Journal: PLoS ONE

doi: 10.1371/journal.pone.0184236

ATPase activity of liposome-reconstituted Pdr11p and Aus1p. Purified Pdr11p and Aus1p were reconstituted into different liposomes (containing Rho-PE as fluorescent lipid marker) and assayed for ATPase activity using [ γ - 32 P] ATP. A: SDS PAGE analysis of a flotation assay of Pdr11p proteoliposomes in a sucrose gradient. Detection of lipids and protein in the same low density fraction validated successful reconstitution. Proteins are visualised by silver staining and lipids by fluorescence from Rho-PE. B: Relative ATPase activity of Pdr11p reconstituted in PS liposomes in presence of the indicated inhibitors: orthovanadate, 1 mM; BeSO 4 , 1 mM; NaF, 5 mM. Data is based on at least two reconstitutions from one purification batch. C: Lipid effect on ATPase activity of reconstituted Pdr11p and Aus1p. All activities are corrected for protein amount in the proteoliposomes. Data is based on two reconstitutions from one purification batch of each protein. PC, PC only; PS, PC/PS (1:1); PG, PC/PG (7:3).
Figure Legend Snippet: ATPase activity of liposome-reconstituted Pdr11p and Aus1p. Purified Pdr11p and Aus1p were reconstituted into different liposomes (containing Rho-PE as fluorescent lipid marker) and assayed for ATPase activity using [ γ - 32 P] ATP. A: SDS PAGE analysis of a flotation assay of Pdr11p proteoliposomes in a sucrose gradient. Detection of lipids and protein in the same low density fraction validated successful reconstitution. Proteins are visualised by silver staining and lipids by fluorescence from Rho-PE. B: Relative ATPase activity of Pdr11p reconstituted in PS liposomes in presence of the indicated inhibitors: orthovanadate, 1 mM; BeSO 4 , 1 mM; NaF, 5 mM. Data is based on at least two reconstitutions from one purification batch. C: Lipid effect on ATPase activity of reconstituted Pdr11p and Aus1p. All activities are corrected for protein amount in the proteoliposomes. Data is based on two reconstitutions from one purification batch of each protein. PC, PC only; PS, PC/PS (1:1); PG, PC/PG (7:3).

Techniques Used: Activity Assay, Purification, Marker, SDS Page, Silver Staining, Fluorescence

ATPase activity of solubilised Pdr11p. ATPase activity of the purified detergent-solubilised transporter was assayed as described under “Materials and Methods” using [ γ - 32 P] ATP. A: ATPase activity as a function of pH. Open and filled circles are data from two independent experiments. Values are normalised with respect to the values at pH 7.2 (open circles) or pH 7.4 (closed circles). The dashed line is included to guide the eye. B: Effect of various inhibitors: NaN 3 , 5 mM; ouabain, 5 mM; BeSO 4 , 1 mM; NaF, 5 mM; AlF 3 , 1 mM; orthovanadate, 1 mM; EDTA, 1 mM. C: ATPase activity as a function of orthovanadate concentration. Fitting of data to a dose-response/activity curve (see Material and methods ) gives IC 50 = 4 ± 2 mM, and a Hill coefficient = 0.8 ± 0.2. Results in B and C are the mean ± S.D. from at least two independent experiments relative to the value obtained for the purified detergent-solubilised protein in the absence of inhibitors (control).
Figure Legend Snippet: ATPase activity of solubilised Pdr11p. ATPase activity of the purified detergent-solubilised transporter was assayed as described under “Materials and Methods” using [ γ - 32 P] ATP. A: ATPase activity as a function of pH. Open and filled circles are data from two independent experiments. Values are normalised with respect to the values at pH 7.2 (open circles) or pH 7.4 (closed circles). The dashed line is included to guide the eye. B: Effect of various inhibitors: NaN 3 , 5 mM; ouabain, 5 mM; BeSO 4 , 1 mM; NaF, 5 mM; AlF 3 , 1 mM; orthovanadate, 1 mM; EDTA, 1 mM. C: ATPase activity as a function of orthovanadate concentration. Fitting of data to a dose-response/activity curve (see Material and methods ) gives IC 50 = 4 ± 2 mM, and a Hill coefficient = 0.8 ± 0.2. Results in B and C are the mean ± S.D. from at least two independent experiments relative to the value obtained for the purified detergent-solubilised protein in the absence of inhibitors (control).

Techniques Used: Activity Assay, Purification, Concentration Assay

28) Product Images from "Phosphorylation of human enhancer filamentation 1 (HEF1) stimulates interaction with Polo-like kinase 1 leading to HEF1 localization to focal adhesions"

Article Title: Phosphorylation of human enhancer filamentation 1 (HEF1) stimulates interaction with Polo-like kinase 1 leading to HEF1 localization to focal adhesions

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M117.802587

CK1δ induces the phosphorylation of Ser-780 and Thr-804 residues on HEF1, leading to the formation of the HEF1–Plk1 complex. A , expression of CK1δ induces the phosphorylation of Ser-780 and Thr-804 residues on HEF1. HEK293T cells co-transfected with FLAG-HEF1 WT and a Myc-empty vector (+ Vector ), Myc-tagged CK1δ (+ Myc-CK1 δ) vector, or CK1ϵ (+ Myc-CK1 ϵ) vector were subjected to an immunoprecipitation assay. Cell lysates were immunoprecipitated with either anti-phospho-Ser-780 or -Thr-804 antiserum, with 10 μg/ml non-phospho-Ser-780 (+ S780 pep ) or non-phospho-Thr-804 (+ T804 pep ) peptide, respectively, and then immunoblotted with an anti-FLAG antibody. Cell lysates were probed with either anti-FLAG or -Myc antibody.  B , expression of CK1δ induces HEF1–Plk1 complex formation. HEK293T cells co-transfected with FLAG-HEF1 T6 and a Myc-empty vector (+ Vector ), Myc-tagged CK1δ (+ Myc-CK1 δ) vector, or CK1ϵ (+ Myc-CK1 ϵ) vector were subjected to a PBD pulldown assay using GST-Plk1 PBD WT. The resulting precipitates were immunoblotted with the indicated antibodies. Note that the expression of CK1δ greatly enhances FLAG-HEF1 T6 binding to Plk1 PBD.  C , CK1δ directly phosphorylates HEF1. The bacterially purified GST-HEF1 WT proteins were reacted with either a bacterially purified GST-CK1δ WT or K38M (kinase-dead mutant) in the presence of [γ- 32 p]ATP, and the resulting samples were then separated by 10% SDS-PAGE and exposed on an X-ray film (Autorad). CBB represents the amount of loaded protein.  Asterisks  indicate degradation products of GST-HEF1 protein.  D , HEF1 S780A/T804A double mutant reduces HEF1 phosphorylation by CK1δ. The bacterially purified GST-CK1δ WT proteins were reacted with either a bacterially purified GST-HEF1 WT or S780A/T804A mutant in the presence of [γ- 32 p]ATP, and the resulting samples were then separated by 10% SDS-PAGE and exposed on an X-ray film (Autorad). CBB represents the amount of loaded protein.  Asterisks  indicate degradation products of GST-HEF1 protein.  E , time course of HEF1 WT and HEF1 S780A/T804A mutant phosphorylation by CK1δ. The kinase reaction was carried out as indicated in  D . A dried SDS-polyacrylamide gel band was excised and dissolved in 30% H 2 O 2 . Phosphorylation (cpm) was measured by liquid scintillation counting. **,  p
Figure Legend Snippet: CK1δ induces the phosphorylation of Ser-780 and Thr-804 residues on HEF1, leading to the formation of the HEF1–Plk1 complex. A , expression of CK1δ induces the phosphorylation of Ser-780 and Thr-804 residues on HEF1. HEK293T cells co-transfected with FLAG-HEF1 WT and a Myc-empty vector (+ Vector ), Myc-tagged CK1δ (+ Myc-CK1 δ) vector, or CK1ϵ (+ Myc-CK1 ϵ) vector were subjected to an immunoprecipitation assay. Cell lysates were immunoprecipitated with either anti-phospho-Ser-780 or -Thr-804 antiserum, with 10 μg/ml non-phospho-Ser-780 (+ S780 pep ) or non-phospho-Thr-804 (+ T804 pep ) peptide, respectively, and then immunoblotted with an anti-FLAG antibody. Cell lysates were probed with either anti-FLAG or -Myc antibody. B , expression of CK1δ induces HEF1–Plk1 complex formation. HEK293T cells co-transfected with FLAG-HEF1 T6 and a Myc-empty vector (+ Vector ), Myc-tagged CK1δ (+ Myc-CK1 δ) vector, or CK1ϵ (+ Myc-CK1 ϵ) vector were subjected to a PBD pulldown assay using GST-Plk1 PBD WT. The resulting precipitates were immunoblotted with the indicated antibodies. Note that the expression of CK1δ greatly enhances FLAG-HEF1 T6 binding to Plk1 PBD. C , CK1δ directly phosphorylates HEF1. The bacterially purified GST-HEF1 WT proteins were reacted with either a bacterially purified GST-CK1δ WT or K38M (kinase-dead mutant) in the presence of [γ- 32 p]ATP, and the resulting samples were then separated by 10% SDS-PAGE and exposed on an X-ray film (Autorad). CBB represents the amount of loaded protein. Asterisks indicate degradation products of GST-HEF1 protein. D , HEF1 S780A/T804A double mutant reduces HEF1 phosphorylation by CK1δ. The bacterially purified GST-CK1δ WT proteins were reacted with either a bacterially purified GST-HEF1 WT or S780A/T804A mutant in the presence of [γ- 32 p]ATP, and the resulting samples were then separated by 10% SDS-PAGE and exposed on an X-ray film (Autorad). CBB represents the amount of loaded protein. Asterisks indicate degradation products of GST-HEF1 protein. E , time course of HEF1 WT and HEF1 S780A/T804A mutant phosphorylation by CK1δ. The kinase reaction was carried out as indicated in D . A dried SDS-polyacrylamide gel band was excised and dissolved in 30% H 2 O 2 . Phosphorylation (cpm) was measured by liquid scintillation counting. **, p

Techniques Used: Expressing, Transfection, Plasmid Preparation, Immunoprecipitation, Binding Assay, Purification, Mutagenesis, SDS Page

29) Product Images from "ML290 is a biased allosteric agonist at the relaxin receptor RXFP1"

Article Title: ML290 is a biased allosteric agonist at the relaxin receptor RXFP1

Journal: Scientific Reports

doi: 10.1038/s41598-017-02916-5

Signal transduction pathways activated by ML290 in human primary vascular cells and in human cardiac fibroblasts (HCF). In ( A ) ML290 (30 min) concentration-dependently increased cAMP accumulation in HCAECs ( , n = 7), HUVECs ( , n = 7), HUASMCs ( , n = 5), HUVSMCs ( , n = 4) but not in HUAECs that do not express cell surface RXFP1 ( , n = 4); in ( B ) ML290 (30 min) also increased cGMP accumulation in HCAECs (n = 7), HUVECs (n = 7), HUASMCs (n = 7), HUVSMCs (n = 4) but not in HUAECs (n = 4); In ( C ) ML290 increased p-p38MAPK (15 min) but only in HUASMC and HUVSMCs (n = 3); In ( D ) ML290 did not cause ERK1/2 phosphorylation in HCAECs (n = 3), HUVECs (n = 3), HUASMCs (n = 3), HUVSMCs (n = 3) or HUAECs (n = 3). In HCFs, H2 relaxin activated cGMP accumulation in a concentration-dependent manner (( E ) n = 5) (40 min) and p-ERK1/2 (( F ) n = 3) (5 min). ML290 did not activate p-ERK1/2 (F; n = 3) but did induce cGMP accumulation (E; n = 5) in a concentration-dependent manner albeit with lower potency than H2 relaxin. Data shown are mean ± SEM of ‘n’ independent experiments.
Figure Legend Snippet: Signal transduction pathways activated by ML290 in human primary vascular cells and in human cardiac fibroblasts (HCF). In ( A ) ML290 (30 min) concentration-dependently increased cAMP accumulation in HCAECs ( , n = 7), HUVECs ( , n = 7), HUASMCs ( , n = 5), HUVSMCs ( , n = 4) but not in HUAECs that do not express cell surface RXFP1 ( , n = 4); in ( B ) ML290 (30 min) also increased cGMP accumulation in HCAECs (n = 7), HUVECs (n = 7), HUASMCs (n = 7), HUVSMCs (n = 4) but not in HUAECs (n = 4); In ( C ) ML290 increased p-p38MAPK (15 min) but only in HUASMC and HUVSMCs (n = 3); In ( D ) ML290 did not cause ERK1/2 phosphorylation in HCAECs (n = 3), HUVECs (n = 3), HUASMCs (n = 3), HUVSMCs (n = 3) or HUAECs (n = 3). In HCFs, H2 relaxin activated cGMP accumulation in a concentration-dependent manner (( E ) n = 5) (40 min) and p-ERK1/2 (( F ) n = 3) (5 min). ML290 did not activate p-ERK1/2 (F; n = 3) but did induce cGMP accumulation (E; n = 5) in a concentration-dependent manner albeit with lower potency than H2 relaxin. Data shown are mean ± SEM of ‘n’ independent experiments.

Techniques Used: Transduction, Concentration Assay

Activation of ERK1/2, p38MAPK and generation of cAMP by H2 relaxin and ML290. In HEK-RXFP1 cells, H2 relaxin activated p-ERK1/2 ( A ) 5 min), p-p38MAPK ( B ) 15 min) and cAMP accumulation ( C ) 30 min) in a concentration-dependent manner. ML290 did not directly activate p-ERK1/2 ( A ), but did activate p38MAPK ( B ) with lower efficacy and cAMP accumulation with similar efficacy but significantly lower potency than H2 relaxin ( C ). 10 min pretreatment with ML290 enhanced p-ERK1/2 activation produced by relaxin ( D ) 4 min). Data are mean ± SEM for 4–8 independent experiments.
Figure Legend Snippet: Activation of ERK1/2, p38MAPK and generation of cAMP by H2 relaxin and ML290. In HEK-RXFP1 cells, H2 relaxin activated p-ERK1/2 ( A ) 5 min), p-p38MAPK ( B ) 15 min) and cAMP accumulation ( C ) 30 min) in a concentration-dependent manner. ML290 did not directly activate p-ERK1/2 ( A ), but did activate p38MAPK ( B ) with lower efficacy and cAMP accumulation with similar efficacy but significantly lower potency than H2 relaxin ( C ). 10 min pretreatment with ML290 enhanced p-ERK1/2 activation produced by relaxin ( D ) 4 min). Data are mean ± SEM for 4–8 independent experiments.

Techniques Used: Activation Assay, Concentration Assay, Produced

30) Product Images from "ML290 is a biased allosteric agonist at the relaxin receptor RXFP1"

Article Title: ML290 is a biased allosteric agonist at the relaxin receptor RXFP1

Journal: Scientific Reports

doi: 10.1038/s41598-017-02916-5

ML290 effects on MMP-2 expression and TGF-β1-induced Smad2 and Smad3 phosphorylation in HCFs. ML290 (1 μM) promoted MMP-2 activity to an equivalent extent to H2 relaxin (0.1 μM) over 72 hours. In ( A upper) a representative cropped zymograph (see Figure S4 ) of duplicate samples from two separate experiments; in ( A lower) mean ± SE OD MMP-2, expressed as the ratio of that of in the untreated control group. **p
Figure Legend Snippet: ML290 effects on MMP-2 expression and TGF-β1-induced Smad2 and Smad3 phosphorylation in HCFs. ML290 (1 μM) promoted MMP-2 activity to an equivalent extent to H2 relaxin (0.1 μM) over 72 hours. In ( A upper) a representative cropped zymograph (see Figure S4 ) of duplicate samples from two separate experiments; in ( A lower) mean ± SE OD MMP-2, expressed as the ratio of that of in the untreated control group. **p

Techniques Used: Expressing, Activity Assay

31) Product Images from "Phosphorylation of β-arrestin2 at Thr383 by MEK underlies β-arrestin-dependent activation of Erk1/2 by GPCRs"

Article Title: Phosphorylation of β-arrestin2 at Thr383 by MEK underlies β-arrestin-dependent activation of Erk1/2 by GPCRs

Journal: eLife

doi: 10.7554/eLife.23777

In vitro phosphorylation of β-arrestin2 versus Erk2 by MEK1. YFP-tagged β-arrestin2 (wild-type or Thr 383 Ala mutant) purified from transfected HEK-293 cells or purified non-activated Erk2 (~1 µg each) were incubated with active MEK1 in presence of [γ- 32 P]-ATP (2 µCi/nmol) for 10 min at 37°C. Proteins were separated by SDS-PAGE and stained with Coomassie colloidal blue (top image) and 32 P incorporation into the different substrates was monitored by autoradiography (bottom image). The data in the histogram, expressed in nmol/min/mg enzyme, represent the means ± SD of 32 P incorporation into β-arrestin2 and Erk2 protein bands in the corresponding experiment after radioactive background subtraction for each lane. DOI: http://dx.doi.org/10.7554/eLife.23777.015 10.7554/eLife.23777.016 This file contains raw values used to build Figure 2—figure supplement 3 . DOI: http://dx.doi.org/10.7554/eLife.23777.016
Figure Legend Snippet: In vitro phosphorylation of β-arrestin2 versus Erk2 by MEK1. YFP-tagged β-arrestin2 (wild-type or Thr 383 Ala mutant) purified from transfected HEK-293 cells or purified non-activated Erk2 (~1 µg each) were incubated with active MEK1 in presence of [γ- 32 P]-ATP (2 µCi/nmol) for 10 min at 37°C. Proteins were separated by SDS-PAGE and stained with Coomassie colloidal blue (top image) and 32 P incorporation into the different substrates was monitored by autoradiography (bottom image). The data in the histogram, expressed in nmol/min/mg enzyme, represent the means ± SD of 32 P incorporation into β-arrestin2 and Erk2 protein bands in the corresponding experiment after radioactive background subtraction for each lane. DOI: http://dx.doi.org/10.7554/eLife.23777.015 10.7554/eLife.23777.016 This file contains raw values used to build Figure 2—figure supplement 3 . DOI: http://dx.doi.org/10.7554/eLife.23777.016

Techniques Used: In Vitro, Mutagenesis, Purification, Transfection, Incubation, SDS Page, Staining, Autoradiography

32) Product Images from "Extended N-terminal region of the essential phosphorelay signaling protein Ypd1 from Cryptococcus neoformans contributes to structural stability, phosphostability and binding of calcium ions"

Article Title: Extended N-terminal region of the essential phosphorelay signaling protein Ypd1 from Cryptococcus neoformans contributes to structural stability, phosphostability and binding of calcium ions

Journal: FEMS Yeast Research

doi: 10.1093/femsyr/fow068

Phosphorylation of CnYpd1 from a heterologous phosphodonor. The HK and RR domains from a heterologous donor, Sln1 from S. cerevisiae (Sln1-HKR1), were used to phosphorylate CnYpd1. ScSln1-HKR1 was autophosphorylated using 0.1 μM γ- 32 P-labeled ATP (lane 1). ScSln1-HKR1 was incubated with CnYpd1 alone (lane 2) or with ScSsk1-R2 (lane 3), CnYpd1-H138Q alone (lane 4) or with ScSsk1-R2 (lane 5).
Figure Legend Snippet: Phosphorylation of CnYpd1 from a heterologous phosphodonor. The HK and RR domains from a heterologous donor, Sln1 from S. cerevisiae (Sln1-HKR1), were used to phosphorylate CnYpd1. ScSln1-HKR1 was autophosphorylated using 0.1 μM γ- 32 P-labeled ATP (lane 1). ScSln1-HKR1 was incubated with CnYpd1 alone (lane 2) or with ScSsk1-R2 (lane 3), CnYpd1-H138Q alone (lane 4) or with ScSsk1-R2 (lane 5).

Techniques Used: Labeling, Incubation

33) Product Images from "Nano-Graphene Oxide Functionalized Bioactive Poly(lactic acid) and Poly(ε-caprolactone) Nanofibrous Scaffolds"

Article Title: Nano-Graphene Oxide Functionalized Bioactive Poly(lactic acid) and Poly(ε-caprolactone) Nanofibrous Scaffolds

Journal: Materials

doi: 10.3390/ma11040566

( a ) FTIR spectra of poly(ε-caprolactone) (PCL), PCL-nGO1, PCL-nGO2.5, and PCL-nGO5 fibers; ( b ) XRD spectra of PCL, PCL-nGO1, PCL-nGO2.5, and PCL-nGO5 fibers; ( c ) SEM images of PCL, PCL-nGO1, PCL-nGO2.5, and PCL-nGO5 fibers.
Figure Legend Snippet: ( a ) FTIR spectra of poly(ε-caprolactone) (PCL), PCL-nGO1, PCL-nGO2.5, and PCL-nGO5 fibers; ( b ) XRD spectra of PCL, PCL-nGO1, PCL-nGO2.5, and PCL-nGO5 fibers; ( c ) SEM images of PCL, PCL-nGO1, PCL-nGO2.5, and PCL-nGO5 fibers.

Techniques Used:

XRD spectra of PLA, PLA-nGO1, PLA-nGO2.5, and PLA-nGO5 fibers.
Figure Legend Snippet: XRD spectra of PLA, PLA-nGO1, PLA-nGO2.5, and PLA-nGO5 fibers.

Techniques Used: Proximity Ligation Assay

( a ) Water contact angle measurements on PLA, PLA-nGO1, PLA-nGO2.5, and PLA-nGO5 fibers; ( b ) The relative cell viability of PLA, PLA-nGO1, PLA-nGO2.5, and PLA-nGO5 fibers; ( c ) Optical microscopy images of PLA-nGO2.5 fibers after 4 days of cell culture; ( d ) SEM image of PLA-nGO2.5 fibers after 4 days of cell culture.
Figure Legend Snippet: ( a ) Water contact angle measurements on PLA, PLA-nGO1, PLA-nGO2.5, and PLA-nGO5 fibers; ( b ) The relative cell viability of PLA, PLA-nGO1, PLA-nGO2.5, and PLA-nGO5 fibers; ( c ) Optical microscopy images of PLA-nGO2.5 fibers after 4 days of cell culture; ( d ) SEM image of PLA-nGO2.5 fibers after 4 days of cell culture.

Techniques Used: Proximity Ligation Assay, Microscopy, Cell Culture

SEM images of PLA, PLA-nGO1, PLA-nGO2.5, and PLA-nGO5 fibers after 11 days of mineralization in simulated body fluid (SBF) and EDS spectra of PLA-nGO5 fibers after 11 days of mineralization in SBF.
Figure Legend Snippet: SEM images of PLA, PLA-nGO1, PLA-nGO2.5, and PLA-nGO5 fibers after 11 days of mineralization in simulated body fluid (SBF) and EDS spectra of PLA-nGO5 fibers after 11 days of mineralization in SBF.

Techniques Used: Proximity Ligation Assay

SEM images of PCL-nGO1, PCL-nGO2.5, and PCL-nGO5 fibers after 11 and 24 days of mineralization in SBF.
Figure Legend Snippet: SEM images of PCL-nGO1, PCL-nGO2.5, and PCL-nGO5 fibers after 11 and 24 days of mineralization in SBF.

Techniques Used:

( a ) TEM images of poly(lactic acid) (PLA) and PLA-nGO1 fibers; ( b ) Fourier transform infrared (FTIR) spectra of PLA, PLA-nGO1, PLA-nGO2.5, and PLA-nGO5 fibers.
Figure Legend Snippet: ( a ) TEM images of poly(lactic acid) (PLA) and PLA-nGO1 fibers; ( b ) Fourier transform infrared (FTIR) spectra of PLA, PLA-nGO1, PLA-nGO2.5, and PLA-nGO5 fibers.

Techniques Used: Transmission Electron Microscopy, Proximity Ligation Assay

( a ) Stress-strain curves of PLA, PLA-nGO1, PLA-nGO2.5 and PLA-nGO5 fibers; ( b ) SEM images of PLA, PLA-nGO1, PLA-nGO2.5, and PLA-nGO5 fibers after tensile break.
Figure Legend Snippet: ( a ) Stress-strain curves of PLA, PLA-nGO1, PLA-nGO2.5 and PLA-nGO5 fibers; ( b ) SEM images of PLA, PLA-nGO1, PLA-nGO2.5, and PLA-nGO5 fibers after tensile break.

Techniques Used: Proximity Ligation Assay

SEM images and the fiber size distribution of PLA, PLA-nGO1, PLA-nGO2.5, and PLA-nGO5 fibers.
Figure Legend Snippet: SEM images and the fiber size distribution of PLA, PLA-nGO1, PLA-nGO2.5, and PLA-nGO5 fibers.

Techniques Used: Proximity Ligation Assay

SEM images of PLA, PLA-nGO1, PLA-nGO2.5, and PLA-nGO5 fibers after 24 days of mineralization in SBF.
Figure Legend Snippet: SEM images of PLA, PLA-nGO1, PLA-nGO2.5, and PLA-nGO5 fibers after 24 days of mineralization in SBF.

Techniques Used: Proximity Ligation Assay

( a ) Stress–strain curves of PCL, PCL-nGO1, PCL-nGO2.5, and PCL-nGO5 fibers; ( b ) SEM images of PCL, PCL-nGO1, PCL-nGO2.5, and PCL-nGO5 fibers after tensile break; ( c ) Optical microscopy images and SEM images of PCL-nGO1 and PCL-nGO2.5 after 4 days of cell culture; ( d ) Water contact angle measurements on PCL, PCL-nGO1, PCL-nGO2.5, and PCL-nGO5 fibers.
Figure Legend Snippet: ( a ) Stress–strain curves of PCL, PCL-nGO1, PCL-nGO2.5, and PCL-nGO5 fibers; ( b ) SEM images of PCL, PCL-nGO1, PCL-nGO2.5, and PCL-nGO5 fibers after tensile break; ( c ) Optical microscopy images and SEM images of PCL-nGO1 and PCL-nGO2.5 after 4 days of cell culture; ( d ) Water contact angle measurements on PCL, PCL-nGO1, PCL-nGO2.5, and PCL-nGO5 fibers.

Techniques Used: Microscopy, Cell Culture

34) Product Images from "Inositol polyphosphate multikinase is a nuclear PI3-kinase with transcriptional regulatory activity"

Article Title: Inositol polyphosphate multikinase is a nuclear PI3-kinase with transcriptional regulatory activity

Journal:

doi: 10.1073/pnas.0506184102

Yeast and mammalian IPMKs are wortmannin-insensitive PI3Ks. Human PI3K p110γ ( A ), rat IPMK ( B ), and yeast IPMK ( C ) were incubated with PI(4,5)P 2 and [γ- 32 P]ATP in the presence of increasing concentrations of wortmannin. Reaction products
Figure Legend Snippet: Yeast and mammalian IPMKs are wortmannin-insensitive PI3Ks. Human PI3K p110γ ( A ), rat IPMK ( B ), and yeast IPMK ( C ) were incubated with PI(4,5)P 2 and [γ- 32 P]ATP in the presence of increasing concentrations of wortmannin. Reaction products

Techniques Used: Incubation

35) Product Images from "Amino Acid Residues within Conserved Domain VI of the Vesicular Stomatitis Virus Large Polymerase Protein Essential for mRNA Cap Methyltransferase Activity"

Article Title: Amino Acid Residues within Conserved Domain VI of the Vesicular Stomatitis Virus Large Polymerase Protein Essential for mRNA Cap Methyltransferase Activity

Journal:

doi: 10.1128/JVI.79.21.13373-13384.2005

Effect of L gene mutations on cap methyltransferase activity. (A) Viral mRNA was synthesized in vitro as described in the text in the presence of either 1 mM SAM or SAH and 15 μCi of [α- 32 P]GTP. Purified mRNAs were digested with 2 U of
Figure Legend Snippet: Effect of L gene mutations on cap methyltransferase activity. (A) Viral mRNA was synthesized in vitro as described in the text in the presence of either 1 mM SAM or SAH and 15 μCi of [α- 32 P]GTP. Purified mRNAs were digested with 2 U of

Techniques Used: Activity Assay, Synthesized, In Vitro, Purification

Transcription of viral mRNAs in vitro. (A) Transcription reactions were performed in vitro in the presence of [α- 32 P]GTP, the RNA was purified and analyzed by electrophoresis on acid-agarose gels as described in Materials and Methods. The products
Figure Legend Snippet: Transcription of viral mRNAs in vitro. (A) Transcription reactions were performed in vitro in the presence of [α- 32 P]GTP, the RNA was purified and analyzed by electrophoresis on acid-agarose gels as described in Materials and Methods. The products

Techniques Used: In Vitro, Purification, Electrophoresis

36) Product Images from "Radiation-generated short DNA fragments may perturb non-homologous end-joining and induce genomic instability"

Article Title: Radiation-generated short DNA fragments may perturb non-homologous end-joining and induce genomic instability

Journal: Journal of radiation research

doi:

DNA-PK kinase activity inhibition by synthesized short DNA fragments. Recombinant p53 protein was incubated with DNA-PK in the presence of γ- 32 P-ATP and various lengths of DNA: synthesized double-stranded oligos (14 mer, 20 mer, 24 mer, 28 mer,
Figure Legend Snippet: DNA-PK kinase activity inhibition by synthesized short DNA fragments. Recombinant p53 protein was incubated with DNA-PK in the presence of γ- 32 P-ATP and various lengths of DNA: synthesized double-stranded oligos (14 mer, 20 mer, 24 mer, 28 mer,

Techniques Used: Activity Assay, Inhibition, Synthesized, Recombinant, Incubation

Comparison of kinase activations stimulated by fragments generated by Co-60 γ-rays and 0.75 MeV fission-neutron irradiation generated short DNA fragments. Recombinant p53 protein was incubated with DNA-PK in the presence of γ- 32 P-ATP and
Figure Legend Snippet: Comparison of kinase activations stimulated by fragments generated by Co-60 γ-rays and 0.75 MeV fission-neutron irradiation generated short DNA fragments. Recombinant p53 protein was incubated with DNA-PK in the presence of γ- 32 P-ATP and

Techniques Used: Generated, Irradiation, Recombinant, Incubation

37) Product Images from "Glucose-Dependent Activation of Bacillus anthracis Toxin Gene Expression and Virulence Requires the Carbon Catabolite Protein CcpA ▿ Toxin Gene Expression and Virulence Requires the Carbon Catabolite Protein CcpA ▿ †"

Article Title: Glucose-Dependent Activation of Bacillus anthracis Toxin Gene Expression and Virulence Requires the Carbon Catabolite Protein CcpA ▿ Toxin Gene Expression and Virulence Requires the Carbon Catabolite Protein CcpA ▿ †

Journal: Journal of Bacteriology

doi: 10.1128/JB.01656-09

Electrophoretic mobility shift assay to determine conditions of CcpA binding to atxA , citZ , and BAS3893 promoter regions. Fragments were generated by PCR amplification and end labeled with [γ- 32 P]ATP via previous phosphorylation with PNK of one
Figure Legend Snippet: Electrophoretic mobility shift assay to determine conditions of CcpA binding to atxA , citZ , and BAS3893 promoter regions. Fragments were generated by PCR amplification and end labeled with [γ- 32 P]ATP via previous phosphorylation with PNK of one

Techniques Used: Electrophoretic Mobility Shift Assay, Binding Assay, Generated, Polymerase Chain Reaction, Amplification, Labeling

38) Product Images from "Human Mitochondrial RNA Polymerase: Evaluation of the Single-Nucleotide-Addition Cycle on Synthetic RNA/DNA Scaffolds"

Article Title: Human Mitochondrial RNA Polymerase: Evaluation of the Single-Nucleotide-Addition Cycle on Synthetic RNA/DNA Scaffolds

Journal: Biochemistry

doi: 10.1021/bi200350d

Characterization of h-mtRNAP-catalyzed pyropho sphorolysis. (a) Experimental design. h-mtRNAP (1 μ ) (0.5 μ M) and [α- 32 P]ATP (0.45 μ M) for 5
Figure Legend Snippet: Characterization of h-mtRNAP-catalyzed pyropho sphorolysis. (a) Experimental design. h-mtRNAP (1 μ ) (0.5 μ M) and [α- 32 P]ATP (0.45 μ M) for 5

Techniques Used:

39) Product Images from "Circadian Autodephosphorylation of Cyanobacterial Clock Protein KaiC Occurs via Formation of ATP as Intermediate *"

Article Title: Circadian Autodephosphorylation of Cyanobacterial Clock Protein KaiC Occurs via Formation of ATP as Intermediate *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M112.350660

Preparation of 32 P-labeled KaiC monomers for autodephosphorylation assays. A , KaiC hexamers were phosphorylated by incubating the samples on ice in the presence of 1 m m [γ- 32 P]ATP. At each time point, an aliquot of the reaction mixture was collected
Figure Legend Snippet: Preparation of 32 P-labeled KaiC monomers for autodephosphorylation assays. A , KaiC hexamers were phosphorylated by incubating the samples on ice in the presence of 1 m m [γ- 32 P]ATP. At each time point, an aliquot of the reaction mixture was collected

Techniques Used: Labeling

40) Product Images from "Colocalization and Interaction of the Porcine Arterivirus Nucleocapsid Protein with the Small Nucleolar RNA-Associated Protein Fibrillarin"

Article Title: Colocalization and Interaction of the Porcine Arterivirus Nucleocapsid Protein with the Small Nucleolar RNA-Associated Protein Fibrillarin

Journal: Journal of Virology

doi: 10.1128/JVI.77.22.12173-12183.2003

Binding of the N protein to the viral genomic RNA. PRRS virions or PRRSV-infected cells were immunoprecipitated using MAb SDOW17 and resolved by SDS-PAGE, followed by transblotting to a nitrocellulose membrane. PRRSV full-length genomic RNA was synthesized in vitro from the full-length cDNA clone of PRRSV using T7 RNA polymerase in the presence of [α- 32 P]UTP. The protein-bound membrane was probed with the radiolabeled RNA transcript for 1 h at 37°C, followed by exposure to a PhosphorImager. (A) Authentic viral proteins. Marc, Marc-145 cells. (B) Recombinant N protein synthesized by T7-based vaccinia virus expression. Lane 4, T7 RNA polymerase expressing recombinant vaccinia virus (vTF7)-infected cells; lane 5, vTF7-infected and pCITE-N gene-transfected cells; lane 6, PRRS virions immunoprecipitated using MAb SDOW17. (C) E. coli- expressed GST fusion proteins. Lane 7, GST alone; lane 8, GST-N fusion protein; lane 9, GST-rotavirus VP8 fusion protein as a negative control.
Figure Legend Snippet: Binding of the N protein to the viral genomic RNA. PRRS virions or PRRSV-infected cells were immunoprecipitated using MAb SDOW17 and resolved by SDS-PAGE, followed by transblotting to a nitrocellulose membrane. PRRSV full-length genomic RNA was synthesized in vitro from the full-length cDNA clone of PRRSV using T7 RNA polymerase in the presence of [α- 32 P]UTP. The protein-bound membrane was probed with the radiolabeled RNA transcript for 1 h at 37°C, followed by exposure to a PhosphorImager. (A) Authentic viral proteins. Marc, Marc-145 cells. (B) Recombinant N protein synthesized by T7-based vaccinia virus expression. Lane 4, T7 RNA polymerase expressing recombinant vaccinia virus (vTF7)-infected cells; lane 5, vTF7-infected and pCITE-N gene-transfected cells; lane 6, PRRS virions immunoprecipitated using MAb SDOW17. (C) E. coli- expressed GST fusion proteins. Lane 7, GST alone; lane 8, GST-N fusion protein; lane 9, GST-rotavirus VP8 fusion protein as a negative control.

Techniques Used: Binding Assay, Infection, Immunoprecipitation, SDS Page, Synthesized, In Vitro, Recombinant, Expressing, Transfection, Negative Control

41) Product Images from "JNK and Ceramide Kinase Govern the Biogenesis of Lipid Droplets through Activation of Group IVA Phospholipase A2 *"

Article Title: JNK and Ceramide Kinase Govern the Biogenesis of Lipid Droplets through Activation of Group IVA Phospholipase A2 *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M109.061515

Cer-1-P and CERK stimulate cPLA 2 α and induce LD biogenesis. A , Western blots of phospho-JNK and phospho-cPLA 2 α from serum-starved CHO-K1 cells that were treated with vehicle, 2.5 μ m C 2 -ceramide, 2.5 μ m Cer-1-P, or 7.5%
Figure Legend Snippet: Cer-1-P and CERK stimulate cPLA 2 α and induce LD biogenesis. A , Western blots of phospho-JNK and phospho-cPLA 2 α from serum-starved CHO-K1 cells that were treated with vehicle, 2.5 μ m C 2 -ceramide, 2.5 μ m Cer-1-P, or 7.5%

Techniques Used: Western Blot

42) Product Images from "Two SERK Receptor-Like Kinases Interact with EMS1 to Control Anther Cell Fate Determination 1Two SERK Receptor-Like Kinases Interact with EMS1 to Control Anther Cell Fate Determination 1 [OPEN]"

Article Title: Two SERK Receptor-Like Kinases Interact with EMS1 to Control Anther Cell Fate Determination 1Two SERK Receptor-Like Kinases Interact with EMS1 to Control Anther Cell Fate Determination 1 [OPEN]

Journal: Plant Physiology

doi: 10.1104/pp.16.01219

In vitro transphosphorylation activities between EMS1 and SERK1/2. A and B, In vitro kinase assays were performed using EMS1-CD, SERK1-CD, and SERK2-CD in the presence of [γ- 32 P]ATP. Top gels, Input proteins stained with Coomassie Brilliant Blue. Bottom gels, Phosphorylation changes analyzed by autoradiography. EMS1-CD T930A and SERK1-CD K330E are inactive forms of EMS1 and SERK1 kinases, respectively. Consistent results were obtained from three independent repeats. C, Identified in vitro autophosphorylation (in black) and transphosphorylation (in blue) sites in the EMS1-CD via mass spectrometry. S, Ser; T, Thr. D, Relative phosphorylation level changes of specific residues in autophosphorylated and transphosphorylated EMS1-CD. Relative phosphorylation was calculated based on the ratio of spectral counts for total versus phosphorylated peptides identified by mass spectrometry analysis.
Figure Legend Snippet: In vitro transphosphorylation activities between EMS1 and SERK1/2. A and B, In vitro kinase assays were performed using EMS1-CD, SERK1-CD, and SERK2-CD in the presence of [γ- 32 P]ATP. Top gels, Input proteins stained with Coomassie Brilliant Blue. Bottom gels, Phosphorylation changes analyzed by autoradiography. EMS1-CD T930A and SERK1-CD K330E are inactive forms of EMS1 and SERK1 kinases, respectively. Consistent results were obtained from three independent repeats. C, Identified in vitro autophosphorylation (in black) and transphosphorylation (in blue) sites in the EMS1-CD via mass spectrometry. S, Ser; T, Thr. D, Relative phosphorylation level changes of specific residues in autophosphorylated and transphosphorylated EMS1-CD. Relative phosphorylation was calculated based on the ratio of spectral counts for total versus phosphorylated peptides identified by mass spectrometry analysis.

Techniques Used: In Vitro, Staining, Autoradiography, Mass Spectrometry

43) Product Images from "Discovery of catalytically active orthologues of the Parkinson's disease kinase PINK1: analysis of substrate specificity and impact of mutations"

Article Title: Discovery of catalytically active orthologues of the Parkinson's disease kinase PINK1: analysis of substrate specificity and impact of mutations

Journal: Open biology

doi: 10.1098/rsob.110012

Effect of Parkinson's disease mutation on PINK1 kinase activity. ( a ) Inset: Schematic of the location of missense PINK1 mutations where the wild-type residue is conserved in both human PINK1 and TcPINK1. Numbering is according to human PINK1. Mutations were introduced into full-length TcPINK1 (1–570), and enzymes (1 µg) were incubated in presence of PINKtide (1 mM) and [γ- 32 P] ATP for 30 min. Reactions were terminated by spotting onto P81 paper, washing in phosphoric acid and quantifying phosphorylation of PINKtide bound to P81 paper. The results are presented as ±s.d. for three experiments undertaken in duplicate. Representative Coomassie-stained gels showing the relative amounts of PINK1 enzyme used for each assay are shown. ( b ) Inset: Schematic of the location of C-terminally truncating PINK1 mutations. Numbering is according to human PINK1. Mutations were introduced into full-length TcPINK1 (1–570), enzymes (1 µg) were incubated in presence of PINKtide (1 mM) and [γ- 32 P] ATP for 30 min. Reactions were terminated by spotting onto P81 paper, washing in phosphoric acid and quantifying phosphorylation of PINKtide bound to P81 paper. The results are presented as ±s.d. for two experiments undertaken in duplicate. Representative Coomassie-stained gels showing the relative amounts of PINK1 enzyme used for each assay are shown.
Figure Legend Snippet: Effect of Parkinson's disease mutation on PINK1 kinase activity. ( a ) Inset: Schematic of the location of missense PINK1 mutations where the wild-type residue is conserved in both human PINK1 and TcPINK1. Numbering is according to human PINK1. Mutations were introduced into full-length TcPINK1 (1–570), and enzymes (1 µg) were incubated in presence of PINKtide (1 mM) and [γ- 32 P] ATP for 30 min. Reactions were terminated by spotting onto P81 paper, washing in phosphoric acid and quantifying phosphorylation of PINKtide bound to P81 paper. The results are presented as ±s.d. for three experiments undertaken in duplicate. Representative Coomassie-stained gels showing the relative amounts of PINK1 enzyme used for each assay are shown. ( b ) Inset: Schematic of the location of C-terminally truncating PINK1 mutations. Numbering is according to human PINK1. Mutations were introduced into full-length TcPINK1 (1–570), enzymes (1 µg) were incubated in presence of PINKtide (1 mM) and [γ- 32 P] ATP for 30 min. Reactions were terminated by spotting onto P81 paper, washing in phosphoric acid and quantifying phosphorylation of PINKtide bound to P81 paper. The results are presented as ±s.d. for two experiments undertaken in duplicate. Representative Coomassie-stained gels showing the relative amounts of PINK1 enzyme used for each assay are shown.

Techniques Used: Mutagenesis, Activity Assay, Incubation, Staining

Characterization of active insect orthologues of PINK1. ( a ) Assessment of activity of wild-type N-terminally truncated human PINK1 (125–581) expressed in E. coli and Sf9 cells, full-length D. melanogaster PINK1 (dPINK1, 1–721), T. castaneum PINK1 (TcPINK1, 1–570) and P. humanus corporis PINK1 (PhcPINK1, 1–575), and corresponding kinase-inactive mutants (HsPINK1-D384A, dPINK1-D501A, TcPINK1-D359A, PhcPINK1-D357A) against myelin basic protein (MBP). The indicated enzymes (1 µg) were incubated in the presence of 5 µg MBP and [γ- 32 P] ATP for 30 min. Reactions were terminated by spotting on P81 paper, washing in phosphoric acid and quantifying phosphorylation of myelin basic protein. The results are presented as ±s.d. for a representative experiment undertaken in duplicate (upper panel). In the lower panel, representative Coomassie-stained gels showing the relative amounts of PINK1 enzyme used for each assay are shown. Fine dividing lines indicate that reactions were resolved on separate gels and grouped in the final figure. ( b ) Assessment of kinase activity of wild-type or kinase inactive (D359A) full-length (1–570), N-terminal truncation (128–570 and 155–570) and N- and C-terminal truncation mutants (155–486) of TcPINK1. The indicated forms of TcPINK1 (1 µg) were incubated in the presence (+) or absence (−) of myelin basic protein (2 µM) and [γ- 32 P] ATP for 30 min. Reactions were terminated by the addition of SDS sample buffer and separated by SDS-PAGE. Gels were analysed by Coomassie staining (upper panel) and incorporation of [γ- 32 P] ATP was detected by autoradiography (lower panel). Fine dividing lines indicate that reactions were resolved on separate gels and grouped in the final figure. ( c ) Analysis of T. castaneum and P. humanus corporis PINK1 function in vivo . TcPINK1 or PhcPINK1 was ectopically expressed in Drosophila lacking endogenous PINK1. Flight ability, climbing ability and presence of thoracic indentations were quantified. Genotypes are as follows. Control: PINK1 B9 /+, mutant: PINK1 B9 /Y; da-GAL4 /+, mutant rescue: PINK1 B9 /Y; da-GAL4 /+, UAS-Tb.PINK1 2a /+ or PINK1 B9 /Y; da-GAL4 /+, UAS-Phc.PINK1 1 /+. Data are presented as mean ± s.e.m.
Figure Legend Snippet: Characterization of active insect orthologues of PINK1. ( a ) Assessment of activity of wild-type N-terminally truncated human PINK1 (125–581) expressed in E. coli and Sf9 cells, full-length D. melanogaster PINK1 (dPINK1, 1–721), T. castaneum PINK1 (TcPINK1, 1–570) and P. humanus corporis PINK1 (PhcPINK1, 1–575), and corresponding kinase-inactive mutants (HsPINK1-D384A, dPINK1-D501A, TcPINK1-D359A, PhcPINK1-D357A) against myelin basic protein (MBP). The indicated enzymes (1 µg) were incubated in the presence of 5 µg MBP and [γ- 32 P] ATP for 30 min. Reactions were terminated by spotting on P81 paper, washing in phosphoric acid and quantifying phosphorylation of myelin basic protein. The results are presented as ±s.d. for a representative experiment undertaken in duplicate (upper panel). In the lower panel, representative Coomassie-stained gels showing the relative amounts of PINK1 enzyme used for each assay are shown. Fine dividing lines indicate that reactions were resolved on separate gels and grouped in the final figure. ( b ) Assessment of kinase activity of wild-type or kinase inactive (D359A) full-length (1–570), N-terminal truncation (128–570 and 155–570) and N- and C-terminal truncation mutants (155–486) of TcPINK1. The indicated forms of TcPINK1 (1 µg) were incubated in the presence (+) or absence (−) of myelin basic protein (2 µM) and [γ- 32 P] ATP for 30 min. Reactions were terminated by the addition of SDS sample buffer and separated by SDS-PAGE. Gels were analysed by Coomassie staining (upper panel) and incorporation of [γ- 32 P] ATP was detected by autoradiography (lower panel). Fine dividing lines indicate that reactions were resolved on separate gels and grouped in the final figure. ( c ) Analysis of T. castaneum and P. humanus corporis PINK1 function in vivo . TcPINK1 or PhcPINK1 was ectopically expressed in Drosophila lacking endogenous PINK1. Flight ability, climbing ability and presence of thoracic indentations were quantified. Genotypes are as follows. Control: PINK1 B9 /+, mutant: PINK1 B9 /Y; da-GAL4 /+, mutant rescue: PINK1 B9 /Y; da-GAL4 /+, UAS-Tb.PINK1 2a /+ or PINK1 B9 /Y; da-GAL4 /+, UAS-Phc.PINK1 1 /+. Data are presented as mean ± s.e.m.

Techniques Used: Activity Assay, Incubation, Staining, SDS Page, Autoradiography, In Vivo, Mutagenesis

44) Product Images from "Protein Kinase A-Dependent Phosphorylation of Serine 119 in the Proto-Oncogenic Serine/Arginine-Rich Splicing Factor 1 Modulates Its Activity as a Splicing Enhancer Protein"

Article Title: Protein Kinase A-Dependent Phosphorylation of Serine 119 in the Proto-Oncogenic Serine/Arginine-Rich Splicing Factor 1 Modulates Its Activity as a Splicing Enhancer Protein

Journal: Genes & Cancer

doi: 10.1177/1947601911430226

Mutation of serine 119 decreases the ability of SRSF1 to interact with RNA. ( A ) 293T cells were transfected with SRSF1 (lanes 1 and 2) or SRSF1 S119A (lanes 3 and 4). Twenty hours posttransfection, the cells were UV cross-linked and lysed. The cell lysates were adjusted to equal protein concentration and treated with T1 RNase before IPs with magnetic beads conjugated with either mouse IgG (lanes 1 and 3) or anti-SRSF1 (lanes 2 and 4). Immunoprecipitated samples were dephosphorylated, labeled with γ-[ 32 P]-ATP by PNK kinase, and run on a denaturating polyacrylamide gel before analysis by autoradiography. The immunoblot (lower panel) shows the amount of SRSF1 in the cell lysate. The arrows indicate accumulation of specific RNA species. ( B ) Lanes in unsaturated images were manually detected in Adobe Photoshop using identical frames. The obtained intensities were adjusted for background and analyzed in GraphPad Prism by a paired t test ( n = 3).
Figure Legend Snippet: Mutation of serine 119 decreases the ability of SRSF1 to interact with RNA. ( A ) 293T cells were transfected with SRSF1 (lanes 1 and 2) or SRSF1 S119A (lanes 3 and 4). Twenty hours posttransfection, the cells were UV cross-linked and lysed. The cell lysates were adjusted to equal protein concentration and treated with T1 RNase before IPs with magnetic beads conjugated with either mouse IgG (lanes 1 and 3) or anti-SRSF1 (lanes 2 and 4). Immunoprecipitated samples were dephosphorylated, labeled with γ-[ 32 P]-ATP by PNK kinase, and run on a denaturating polyacrylamide gel before analysis by autoradiography. The immunoblot (lower panel) shows the amount of SRSF1 in the cell lysate. The arrows indicate accumulation of specific RNA species. ( B ) Lanes in unsaturated images were manually detected in Adobe Photoshop using identical frames. The obtained intensities were adjusted for background and analyzed in GraphPad Prism by a paired t test ( n = 3).

Techniques Used: Mutagenesis, Transfection, Protein Concentration, Magnetic Beads, Immunoprecipitation, Labeling, Autoradiography

PKA phosphorylates SRSF1 at serine 119 in vitro . Purified SRSF1 (lanes 1 and 2), SRSF1 S119A (lanes 3 and 4), SRSF1 ΔRS (lanes 5 and 6), and SRSF1 ΔRS S119A (lanes 7 and 8) were incubated with active or heat-inactivated PKA Cα1 and γ-[ 32 P]-ATP in a reaction buffer. The samples were analyzed by SDS-PAGE followed by Coomassie staining (lower panel) and autoradiography (upper panel).
Figure Legend Snippet: PKA phosphorylates SRSF1 at serine 119 in vitro . Purified SRSF1 (lanes 1 and 2), SRSF1 S119A (lanes 3 and 4), SRSF1 ΔRS (lanes 5 and 6), and SRSF1 ΔRS S119A (lanes 7 and 8) were incubated with active or heat-inactivated PKA Cα1 and γ-[ 32 P]-ATP in a reaction buffer. The samples were analyzed by SDS-PAGE followed by Coomassie staining (lower panel) and autoradiography (upper panel).

Techniques Used: In Vitro, Purification, Incubation, SDS Page, Staining, Autoradiography

45) Product Images from "The Rev1 interacting region (RIR) motif in the scaffold protein XRCC1 mediates a low-affinity interaction with polynucleotide kinase/phosphatase (PNKP) during DNA single-strand break repair"

Article Title: The Rev1 interacting region (RIR) motif in the scaffold protein XRCC1 mediates a low-affinity interaction with polynucleotide kinase/phosphatase (PNKP) during DNA single-strand break repair

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M117.806638

The conserved XRCC1 phenylalanine motif is required for the phosphorylation-independent stimulation of PNKP activity. A , stimulation of PNKP DNA kinase activity. 0.5 μ m PNKP was incubated in the presence of [γ- 32 P]ATP with 10 μ m oligonucleotide substrate and 4 μ m XRCC1-His, XRCC1-His FFF , His-XRCC1 161–406 , or His-XRCC1 161–406-RK for 2 min at 37 °C. The amount of radiolabeled 5′-phosphorylated 24-mer oligonucleotide was then quantified by gel electrophoresis and autoradiography. Data are the mean ± S.D. of three independent experiments. B , stimulation of PNKP DNA kinase enzyme-product turnover. DNA kinase reactions (50 μl) containing 2 μ m 1-nt gapped oligonucleotide substrate and 0.2 μ m PNKP were conducted as above in the absence of XRCC1 for 20 min and XRCC1-His or XRCC1-His FFF then added to 0.8 μ m for a further 20 min. Phosphorylated oligonucleotide product was quantified at the indicated times, as above. Data are the mean ± S.D. of three independent experiments. C , stimulation of PNKP DNA phosphatase activity. DNA phosphatase reactions (30 μl) containing 0.33 μ m 1-nt gapped oligonucleotide substrate and 0.86 μ m PNKP were incubated in the absence of any XRCC1 protein for 20 min and then, where indicated, in the additional presence of 1.65 μ m XRCC1-His or XRCC1-His FFF for a further 20 min. 3′-Dephosphorylated oligonucleotide product was quantified at the indicated times, as above. Data are the mean ± S.D. of three independent experiments. D , Interaction of XRCC1-His (■) and XRCC1-His FFF (●) with 1-nt gapped DNA. Proteins (30 n m ) were excited at 295 nm and the fluorescence intensity at 340 nm was monitored as a function of added 1 nt-gapped DNA substrate (see inset for data with XRCC1-His). The fraction bound, i.e. relative fluorescence ( Rel. Fluor .), versus ligand concentration is plotted.
Figure Legend Snippet: The conserved XRCC1 phenylalanine motif is required for the phosphorylation-independent stimulation of PNKP activity. A , stimulation of PNKP DNA kinase activity. 0.5 μ m PNKP was incubated in the presence of [γ- 32 P]ATP with 10 μ m oligonucleotide substrate and 4 μ m XRCC1-His, XRCC1-His FFF , His-XRCC1 161–406 , or His-XRCC1 161–406-RK for 2 min at 37 °C. The amount of radiolabeled 5′-phosphorylated 24-mer oligonucleotide was then quantified by gel electrophoresis and autoradiography. Data are the mean ± S.D. of three independent experiments. B , stimulation of PNKP DNA kinase enzyme-product turnover. DNA kinase reactions (50 μl) containing 2 μ m 1-nt gapped oligonucleotide substrate and 0.2 μ m PNKP were conducted as above in the absence of XRCC1 for 20 min and XRCC1-His or XRCC1-His FFF then added to 0.8 μ m for a further 20 min. Phosphorylated oligonucleotide product was quantified at the indicated times, as above. Data are the mean ± S.D. of three independent experiments. C , stimulation of PNKP DNA phosphatase activity. DNA phosphatase reactions (30 μl) containing 0.33 μ m 1-nt gapped oligonucleotide substrate and 0.86 μ m PNKP were incubated in the absence of any XRCC1 protein for 20 min and then, where indicated, in the additional presence of 1.65 μ m XRCC1-His or XRCC1-His FFF for a further 20 min. 3′-Dephosphorylated oligonucleotide product was quantified at the indicated times, as above. Data are the mean ± S.D. of three independent experiments. D , Interaction of XRCC1-His (■) and XRCC1-His FFF (●) with 1-nt gapped DNA. Proteins (30 n m ) were excited at 295 nm and the fluorescence intensity at 340 nm was monitored as a function of added 1 nt-gapped DNA substrate (see inset for data with XRCC1-His). The fraction bound, i.e. relative fluorescence ( Rel. Fluor .), versus ligand concentration is plotted.

Techniques Used: Activity Assay, Incubation, Field Flow Fractionation, Nucleic Acid Electrophoresis, Autoradiography, Fluorescence, Concentration Assay

46) Product Images from "Transcriptional Corepressors HIPK1 and HIPK2 Control Angiogenesis Via TGF-?-TAK1-Dependent Mechanism"

Article Title: Transcriptional Corepressors HIPK1 and HIPK2 Control Angiogenesis Via TGF-?-TAK1-Dependent Mechanism

Journal: PLoS Biology

doi: 10.1371/journal.pbio.1001527

TGF-β activates HIPK2 by phosphorylating a highly conserved tyrosine residue on position 361. (A) Amino acid sequence alignment of the HIPK protein family from human and mouse reveals a stretch of highly conserved residues from position 346 to 371 in the activation segment of the subdomain VII in HIPK2. (B) Alignment of the similar regions of HIPK2 (346 to 371) from different species confirms that these amino acid residues are highly conserved from nematodes to the vertebrates. Conserved amino acids that can potentially be phosphorylated in MAPK signaling pathway are shown in bold. (C) The combined immunoprecipitation and in vitro kinase (IP-IVK) assays show that TGF-β treatment promotes the ability of wild-type HIPK2 to incorporate γ- 32 P-ATP. In contrast, kinase inactive HIPK2-K221A fails to incorporate γ- 32 P-ATP. While HIPK2-S359A and HIPK2-T360A mutant proteins can still incorporate γ- 32 P-ATP in response to TGF-β treatment, the Y361F mutation in HIPK2 completely eliminates its ability to incorporate γ- 32 P-ATP. (D) TGF-β and TAK1-induced phosphorylation of HIPK2 occurs primarily on Y361 residue in HIPK2. HIPK2-Y361F mutant completely loses its ability to incorporate γ- 32 P-ATP upon activation by TGF-β or TAK1. Data are shown as mean + s.e.m., n = 3. Statistics in (C) and (D) use Student's t test. * p
Figure Legend Snippet: TGF-β activates HIPK2 by phosphorylating a highly conserved tyrosine residue on position 361. (A) Amino acid sequence alignment of the HIPK protein family from human and mouse reveals a stretch of highly conserved residues from position 346 to 371 in the activation segment of the subdomain VII in HIPK2. (B) Alignment of the similar regions of HIPK2 (346 to 371) from different species confirms that these amino acid residues are highly conserved from nematodes to the vertebrates. Conserved amino acids that can potentially be phosphorylated in MAPK signaling pathway are shown in bold. (C) The combined immunoprecipitation and in vitro kinase (IP-IVK) assays show that TGF-β treatment promotes the ability of wild-type HIPK2 to incorporate γ- 32 P-ATP. In contrast, kinase inactive HIPK2-K221A fails to incorporate γ- 32 P-ATP. While HIPK2-S359A and HIPK2-T360A mutant proteins can still incorporate γ- 32 P-ATP in response to TGF-β treatment, the Y361F mutation in HIPK2 completely eliminates its ability to incorporate γ- 32 P-ATP. (D) TGF-β and TAK1-induced phosphorylation of HIPK2 occurs primarily on Y361 residue in HIPK2. HIPK2-Y361F mutant completely loses its ability to incorporate γ- 32 P-ATP upon activation by TGF-β or TAK1. Data are shown as mean + s.e.m., n = 3. Statistics in (C) and (D) use Student's t test. * p

Techniques Used: Sequencing, Activation Assay, Immunoprecipitation, In Vitro, Mutagenesis

TGF-β–TAK1 promotes HIPK2 activity through protein–protein interaction and protects HIPK2 from proteasome-mediated degradation. (A) TGF-β promotes HIPK2 kinase activity in HEK293T cells, whereas kinase inactive HIPK2-K221A shows no incorporation of γ- 32 P-ATP upon TGF-β treatment. (B) The ability of TGF-β to activate HIPK2 kinase activity can be blocked by TGF-β type I receptor inhibitor SB431542. (C and D) TGF-β and wild-type TAK1 activate HIPK2 kinase and maintain the stability of HIPK2 protein. In contrast, dominant negative TAK1 (DN-TAK1) promotes HIPK2 degradation via the proteasome pathway.
Figure Legend Snippet: TGF-β–TAK1 promotes HIPK2 activity through protein–protein interaction and protects HIPK2 from proteasome-mediated degradation. (A) TGF-β promotes HIPK2 kinase activity in HEK293T cells, whereas kinase inactive HIPK2-K221A shows no incorporation of γ- 32 P-ATP upon TGF-β treatment. (B) The ability of TGF-β to activate HIPK2 kinase activity can be blocked by TGF-β type I receptor inhibitor SB431542. (C and D) TGF-β and wild-type TAK1 activate HIPK2 kinase and maintain the stability of HIPK2 protein. In contrast, dominant negative TAK1 (DN-TAK1) promotes HIPK2 degradation via the proteasome pathway.

Techniques Used: Activity Assay, Dominant Negative Mutation

47) Product Images from "DNA-PK Target Identification Reveals Novel Links between DNA Repair Signaling and Cytoskeletal Regulation"

Article Title: DNA-PK Target Identification Reveals Novel Links between DNA Repair Signaling and Cytoskeletal Regulation

Journal: PLoS ONE

doi: 10.1371/journal.pone.0080313

Differential phosphorylation pattern and total protein status after Dbait32Hc treatment. (A) SDS-polyacrylamide gel electrophoresis after isoelectric focusing (pH range 4.5–5.5) of MRC-5 lysates treated with Dbait32Hc or 8H. Total protein was detected by Sypro Ruby (red, SR) staining and phosphorylation was monitored by Pro-Q Diamond (green, Pro-Q) staining of the same gel. Spots displaying a marked increase in phosphorylation after treatment are highlighted (white arrows). (B) Higher magnification of selected spots from (A) showing higher levels of phosphorylation (arrows) of the indicated proteins after Dbait32Hc treatment than after transfection with the control, 8H. No difference in total protein levels was founds. Proteins displaying at least a 10-fold increase in Pro-Q Diamond staining that could be unambiguously assigned to proteins stained by Sypro Ruby were excised and analyzed by LC-MS/MS. (C) In vitro phosphorylation of vimentin by DNA-PK. Purified DNA-PK (DNA-PKcs and Ku) was incubated with [γ- 32 P]ATP and the indicated amounts of purified vimentin protein. Dbait32Hc was added where indicated, to activate DNA-PK, and the proteins were then denatured, separated by SDS-polyacrylamide gel electrophoresis and analyzed by autoradiography. (D) Peptides and phosphosites of in vitro DNA-PK-phosphorylated vimentin, as identified by LC-MS/MS with the LTQ-Orbitrap after trypsin digestion. (pT) and (pS) correspond to phosphorylated threonine and serine, respectively.
Figure Legend Snippet: Differential phosphorylation pattern and total protein status after Dbait32Hc treatment. (A) SDS-polyacrylamide gel electrophoresis after isoelectric focusing (pH range 4.5–5.5) of MRC-5 lysates treated with Dbait32Hc or 8H. Total protein was detected by Sypro Ruby (red, SR) staining and phosphorylation was monitored by Pro-Q Diamond (green, Pro-Q) staining of the same gel. Spots displaying a marked increase in phosphorylation after treatment are highlighted (white arrows). (B) Higher magnification of selected spots from (A) showing higher levels of phosphorylation (arrows) of the indicated proteins after Dbait32Hc treatment than after transfection with the control, 8H. No difference in total protein levels was founds. Proteins displaying at least a 10-fold increase in Pro-Q Diamond staining that could be unambiguously assigned to proteins stained by Sypro Ruby were excised and analyzed by LC-MS/MS. (C) In vitro phosphorylation of vimentin by DNA-PK. Purified DNA-PK (DNA-PKcs and Ku) was incubated with [γ- 32 P]ATP and the indicated amounts of purified vimentin protein. Dbait32Hc was added where indicated, to activate DNA-PK, and the proteins were then denatured, separated by SDS-polyacrylamide gel electrophoresis and analyzed by autoradiography. (D) Peptides and phosphosites of in vitro DNA-PK-phosphorylated vimentin, as identified by LC-MS/MS with the LTQ-Orbitrap after trypsin digestion. (pT) and (pS) correspond to phosphorylated threonine and serine, respectively.

Techniques Used: Polyacrylamide Gel Electrophoresis, Staining, Transfection, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, In Vitro, Purification, Incubation, Autoradiography

48) Product Images from "The CroRS Two-Component Regulatory System Is Required for Intrinsic ?-Lactam Resistance in Enterococcus faecalis"

Article Title: The CroRS Two-Component Regulatory System Is Required for Intrinsic ?-Lactam Resistance in Enterococcus faecalis

Journal: Journal of Bacteriology

doi: 10.1128/JB.185.24.7184-7192.2003

Phosphotransfer reactions catalyzed by CroS S and CroR H . (A) Kinetics of CroS S autophosphorylation. CroS S was incubated with [γ 32 -P]ATP for 0, 5, 10, 30, and 60 min (lanes 1 to 5, respectively) and applied to an SDS-13.5% polyacrylamide gel. (B) Transfer of the phosphate group from the phosphorylated form of CroS S (P-CroS) to CroR H . Phospho-CroS S was prepared (lane 1) and incubated with CroR H for 2, 5, and 20 min (lanes 2, 3, and 4, respectively).
Figure Legend Snippet: Phosphotransfer reactions catalyzed by CroS S and CroR H . (A) Kinetics of CroS S autophosphorylation. CroS S was incubated with [γ 32 -P]ATP for 0, 5, 10, 30, and 60 min (lanes 1 to 5, respectively) and applied to an SDS-13.5% polyacrylamide gel. (B) Transfer of the phosphate group from the phosphorylated form of CroS S (P-CroS) to CroR H . Phospho-CroS S was prepared (lane 1) and incubated with CroR H for 2, 5, and 20 min (lanes 2, 3, and 4, respectively).

Techniques Used: Incubation

49) Product Images from "Functional Analysis of the Mycobacterium tuberculosis MprAB Two-Component Signal Transduction System "

Article Title: Functional Analysis of the Mycobacterium tuberculosis MprAB Two-Component Signal Transduction System

Journal: Infection and Immunity

doi: 10.1128/IAI.71.12.6962-6970.2003

Transphosphorylation between GST-cMprB and M. tuberculosis response regulators. Wild-type GST-cMprB was autophosporylated with [γ- 32 P]ATP and then incubated in the absence of other proteins (lanes 1 and 2) or in the presence of wild-type His-MprA (lanes 3 to 7), the His-MprA (Asp48-Ala) mutant (lanes 8 to 12), or wild-type His-MtrA (lanes 13 to 17). Transphosphorylation reactions were allowed to proceed for 0 min (lanes 1, 3, 8, and 13), 5 min (lanes 4, 9, and 14), 10 min (lanes 5, 10, and 15), 20 min (lanes 6, 11, and 16), or 30 min (lanes 2, 7, 12, and 17). Closed arrows indicate the locations of full-length GST-cMprB and response regulator proteins. Open arrows indicate the locations of truncated (trunc.) forms of GST-cMprB. The asterisk indicates the position of phosphorylated His-MprA species. Transfer of radiolabel from GST-cMprB to response regulator proteins was detected by autoradiography. WT, wild type.
Figure Legend Snippet: Transphosphorylation between GST-cMprB and M. tuberculosis response regulators. Wild-type GST-cMprB was autophosporylated with [γ- 32 P]ATP and then incubated in the absence of other proteins (lanes 1 and 2) or in the presence of wild-type His-MprA (lanes 3 to 7), the His-MprA (Asp48-Ala) mutant (lanes 8 to 12), or wild-type His-MtrA (lanes 13 to 17). Transphosphorylation reactions were allowed to proceed for 0 min (lanes 1, 3, 8, and 13), 5 min (lanes 4, 9, and 14), 10 min (lanes 5, 10, and 15), 20 min (lanes 6, 11, and 16), or 30 min (lanes 2, 7, 12, and 17). Closed arrows indicate the locations of full-length GST-cMprB and response regulator proteins. Open arrows indicate the locations of truncated (trunc.) forms of GST-cMprB. The asterisk indicates the position of phosphorylated His-MprA species. Transfer of radiolabel from GST-cMprB to response regulator proteins was detected by autoradiography. WT, wild type.

Techniques Used: Incubation, Mutagenesis, Autoradiography

In vitro autophosphorylation of GST-cMprB derivatives. (A) Purified GST-cMprB was incubated in the presence of [γ- 32 P]ATP (lanes 1, 3, 5, and 7) or [α- 32 P]ATP (lanes 2, 4, 6, and 8) and divalent cations including Mg 2+ (lanes 1 and 2), Mn 2+ (lanes 3 and 4), and Ca 2+ (lanes 5 and 6) or in the absence of metal (lanes 7 and 8). (B) Wild-type GST-cMprB (lanes 1 to 4) or the GST-cMprB (His249-Gln) mutant (lanes 5 to 8) was incubated in the presence of [γ- 32 P]ATP and Mg 2+ (lanes 1 and 5), Mn 2+ (lanes 2 and 6), or Ca 2+ (lanes 3 and 7) or in the absence of divalent cations (lanes 4 and 8). Phosphorylation of wild-type or mutant GST-cMprB was detected by autoradiography, and polyclonal antibody directed against cMprB was used in Western blotting to confirm similar loading amounts between reactions.
Figure Legend Snippet: In vitro autophosphorylation of GST-cMprB derivatives. (A) Purified GST-cMprB was incubated in the presence of [γ- 32 P]ATP (lanes 1, 3, 5, and 7) or [α- 32 P]ATP (lanes 2, 4, 6, and 8) and divalent cations including Mg 2+ (lanes 1 and 2), Mn 2+ (lanes 3 and 4), and Ca 2+ (lanes 5 and 6) or in the absence of metal (lanes 7 and 8). (B) Wild-type GST-cMprB (lanes 1 to 4) or the GST-cMprB (His249-Gln) mutant (lanes 5 to 8) was incubated in the presence of [γ- 32 P]ATP and Mg 2+ (lanes 1 and 5), Mn 2+ (lanes 2 and 6), or Ca 2+ (lanes 3 and 7) or in the absence of divalent cations (lanes 4 and 8). Phosphorylation of wild-type or mutant GST-cMprB was detected by autoradiography, and polyclonal antibody directed against cMprB was used in Western blotting to confirm similar loading amounts between reactions.

Techniques Used: In Vitro, Purification, Incubation, Mutagenesis, Autoradiography, Western Blot

50) Product Images from "Quantitative Analysis of Dynamic Protein Interactions during Transcription Reveals a Role for Casein Kinase II in Polymerase-associated Factor (PAF) Complex Phosphorylation and Regulation of Histone H2B Monoubiquitylation *"

Article Title: Quantitative Analysis of Dynamic Protein Interactions during Transcription Reveals a Role for Casein Kinase II in Polymerase-associated Factor (PAF) Complex Phosphorylation and Regulation of Histone H2B Monoubiquitylation *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M116.727735

In vitro phosphorylation of FACT and PAF-C by CKII. A , autoradiograph of a representative set of in vitro reactions performed with [γ 32 P]ATP in the presence or absence of Ctr9-FLAG, Spt16-TAP, or recombinant CKII, as indicated at the top . The
Figure Legend Snippet: In vitro phosphorylation of FACT and PAF-C by CKII. A , autoradiograph of a representative set of in vitro reactions performed with [γ 32 P]ATP in the presence or absence of Ctr9-FLAG, Spt16-TAP, or recombinant CKII, as indicated at the top . The

Techniques Used: In Vitro, Autoradiography, Recombinant

51) Product Images from "The role of Nedd4-1 WW domains in binding and regulating human organic anion transporter 1"

Article Title: The role of Nedd4-1 WW domains in binding and regulating human organic anion transporter 1

Journal: American Journal of Physiology - Renal Physiology

doi: 10.1152/ajprenal.00153.2016

Effect of Nedd4-1 on hOAT1 transport activity and kinetics. A : hOAT1-expressing COS-7 cells were transfected with cDNAs for wild-type Nedd4-1 or for the ubiquitin ligase-dead mutant Nedd4-1/C867S. Three-minute uptake of p -aminohippuric acid ([ 3 H]PAH;
Figure Legend Snippet: Effect of Nedd4-1 on hOAT1 transport activity and kinetics. A : hOAT1-expressing COS-7 cells were transfected with cDNAs for wild-type Nedd4-1 or for the ubiquitin ligase-dead mutant Nedd4-1/C867S. Three-minute uptake of p -aminohippuric acid ([ 3 H]PAH;

Techniques Used: Activity Assay, Expressing, Transfection, Mutagenesis

52) Product Images from "Functional Relationship of ATP Hydrolysis, Presynaptic Filament Stability, and Homologous DNA Pairing Activity of the Human Meiotic Recombinase DMC1 *"

Article Title: Functional Relationship of ATP Hydrolysis, Presynaptic Filament Stability, and Homologous DNA Pairing Activity of the Human Meiotic Recombinase DMC1 *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M115.666289

ATP binding and hydrolysis by DMC1 and mutant proteins. A , schematic of the UV cross-linking analysis. DMC1 and mutant proteins were incubated with [γ- 32 P]ATP. Following UV cross-linking, radiolabeled proteins were run on a 13.5% denaturing polyacrylamide
Figure Legend Snippet: ATP binding and hydrolysis by DMC1 and mutant proteins. A , schematic of the UV cross-linking analysis. DMC1 and mutant proteins were incubated with [γ- 32 P]ATP. Following UV cross-linking, radiolabeled proteins were run on a 13.5% denaturing polyacrylamide

Techniques Used: Binding Assay, Mutagenesis, Incubation

53) Product Images from "Poly(ADP-ribose) Polymerase 1 (PARP1) Associates with E3 Ubiquitin-Protein Ligase UHRF1 and Modulates UHRF1 Biological Functions *"

Article Title: Poly(ADP-ribose) Polymerase 1 (PARP1) Associates with E3 Ubiquitin-Protein Ligase UHRF1 and Modulates UHRF1 Biological Functions *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M113.527424

UHRF1 is PARylated preferentially onto its SRA domain but also onto its TTD. A , PARylation of full-length UHRF1 by PARP1. Myc-UHRF1 ( lanes 1 , 2 , 5 , and 6 ) or Myc-TRF2 ( lanes 3 , 4 , 7 , and 8 ) was expressed in COS-1 cells, immunopurified with an anti-Myc antibody, and incubated together with either PARP1 or without PARPs in activity buffer containing [α- 32 P]NAD + and fragmented DNA. Right panel , autoradiography. Left panel , analysis of the fusion proteins with an anti-Myc antibody by Western blotting. B , upper panel , schematic representation of UHRF1 indicating the PARylated TTD and SRA domain. Lower panel , GFP fusion proteins expressing GFP alone ( lanes 1 and 7 ) or different domains of UHRF1 (GFP-Ring (lanes 2 and 8 ), GFP-SRA ( lanes 3 and 9 ), GFP-PHD ( lanes 4 and 10 ), GFP-Ubl ( lanes 5 and 11 ), and GFP-TTD ( lanes 6 and 12 )) were expressed in COS-1 cells, immunopurified by GFP trapping, and incubated together with PARP1 in the activity buffer containing [α-32]PNAD + and fragmented DNA as in A. Left , analysis of the PARylated domains by autoradiography. The upper band represents PARylated PARP1. Right , analysis of the fusion proteins with an anti-GFP antibody by Western blotting.
Figure Legend Snippet: UHRF1 is PARylated preferentially onto its SRA domain but also onto its TTD. A , PARylation of full-length UHRF1 by PARP1. Myc-UHRF1 ( lanes 1 , 2 , 5 , and 6 ) or Myc-TRF2 ( lanes 3 , 4 , 7 , and 8 ) was expressed in COS-1 cells, immunopurified with an anti-Myc antibody, and incubated together with either PARP1 or without PARPs in activity buffer containing [α- 32 P]NAD + and fragmented DNA. Right panel , autoradiography. Left panel , analysis of the fusion proteins with an anti-Myc antibody by Western blotting. B , upper panel , schematic representation of UHRF1 indicating the PARylated TTD and SRA domain. Lower panel , GFP fusion proteins expressing GFP alone ( lanes 1 and 7 ) or different domains of UHRF1 (GFP-Ring (lanes 2 and 8 ), GFP-SRA ( lanes 3 and 9 ), GFP-PHD ( lanes 4 and 10 ), GFP-Ubl ( lanes 5 and 11 ), and GFP-TTD ( lanes 6 and 12 )) were expressed in COS-1 cells, immunopurified by GFP trapping, and incubated together with PARP1 in the activity buffer containing [α-32]PNAD + and fragmented DNA as in A. Left , analysis of the PARylated domains by autoradiography. The upper band represents PARylated PARP1. Right , analysis of the fusion proteins with an anti-GFP antibody by Western blotting.

Techniques Used: Incubation, Activity Assay, Autoradiography, Western Blot, Expressing

54) Product Images from "Characterization of the AtsR Hybrid Sensor Kinase Phosphorelay Pathway and Identification of Its Response Regulator in Burkholderia cenocepacia *"

Article Title: Characterization of the AtsR Hybrid Sensor Kinase Phosphorelay Pathway and Identification of Its Response Regulator in Burkholderia cenocepacia *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M113.489914

Kinetics of phosphotransfer from AtsR to AtsT. A , 5 μmol of AtsR were preincubated with 5 μCi ([γ- 33 P]ATP) in a standard phosphorylation mixture (100 m m Tris-HCl, pH 8, 50 m m KCl, 5 m m MgCl 2 , 1 m m DTT) for 15 min, and then 5 μmol
Figure Legend Snippet: Kinetics of phosphotransfer from AtsR to AtsT. A , 5 μmol of AtsR were preincubated with 5 μCi ([γ- 33 P]ATP) in a standard phosphorylation mixture (100 m m Tris-HCl, pH 8, 50 m m KCl, 5 m m MgCl 2 , 1 m m DTT) for 15 min, and then 5 μmol

Techniques Used:

55) Product Images from "RNA as a Boiling-Resistant Anionic Polymer Material To Build Robust Structures with Defined Shape and Stoichiometry"

Article Title: RNA as a Boiling-Resistant Anionic Polymer Material To Build Robust Structures with Defined Shape and Stoichiometry

Journal: ACS Nano

doi: 10.1021/nn5006254

Boiling temperature and urea resistance assay. (A) The boiling temperature effects on stability of RNA, hybrid, and 2′F-RNA triangles evaluated by 6% native PAGEs. Fixed concentrations of 32 P ATP-labeled [rA*, rB, rC, rD], [rA*, rB, rC, 2′F-D], and [2′F-A*, 2′F–B, 2′F-C, 2′F-D] triangles were incubated with unlabeled rA or 2′F-A strands at ratios from 1:0 to 1:3 for 5 min at 100 °C . The quantified fractions of remaining triangles obtained from three independent experiments are shown on bottom left. (B) Effect of 8 M UREA on stability of triangles. The 8% polyacrylamide gel was cast in 1× TB buffer, and the RNA bands were visualized by total RNA stain in E.B.
Figure Legend Snippet: Boiling temperature and urea resistance assay. (A) The boiling temperature effects on stability of RNA, hybrid, and 2′F-RNA triangles evaluated by 6% native PAGEs. Fixed concentrations of 32 P ATP-labeled [rA*, rB, rC, rD], [rA*, rB, rC, 2′F-D], and [2′F-A*, 2′F–B, 2′F-C, 2′F-D] triangles were incubated with unlabeled rA or 2′F-A strands at ratios from 1:0 to 1:3 for 5 min at 100 °C . The quantified fractions of remaining triangles obtained from three independent experiments are shown on bottom left. (B) Effect of 8 M UREA on stability of triangles. The 8% polyacrylamide gel was cast in 1× TB buffer, and the RNA bands were visualized by total RNA stain in E.B.

Techniques Used: Labeling, Incubation, Staining

56) Product Images from "Comparing the effects of nano-sized sugarcane fiber with cellulose and psyllium on hepatic cellular signaling in mice"

Article Title: Comparing the effects of nano-sized sugarcane fiber with cellulose and psyllium on hepatic cellular signaling in mice

Journal: International Journal of Nanomedicine

doi: 10.2147/IJN.S30887

Effects of dietary fibers on insulin signaling protein abundance and phosphatidylinositol 3 kinase (PI 3K) activity in mice livers. Liver lysates were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and insulin signaling pathway proteins were detected with corresponding specific antibodies as shown in the figures. ( A ) shows that phosphorylation of antiphosphotyrosine 20 (PY 20), insulin receptor substrate (IRS) 1, insulin receptor beta (IR β), and Akt1 were normalized by their corresponding protein contents and PY 20 was normalized by β-actin. ( B ) Hepatic IRS-1-associated PI 3K activity in mice. Liver lysates from basal and insulin-stimulated mice were immunoprecipitated with IRS-1 antibody and protein A agarose. The immune complexes were incubated with reaction buffer containing [γ- 32 P]adenosine 5′-triphosphate, MgCl 2 , MnCl 2 , and phosphatidylinositol for 20 minutes. Autoradiograph was performed after thin-layer chromatography. Notes:  Data presented as mean ± standard error of the mean (n = 9/group). * P
Figure Legend Snippet: Effects of dietary fibers on insulin signaling protein abundance and phosphatidylinositol 3 kinase (PI 3K) activity in mice livers. Liver lysates were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and insulin signaling pathway proteins were detected with corresponding specific antibodies as shown in the figures. ( A ) shows that phosphorylation of antiphosphotyrosine 20 (PY 20), insulin receptor substrate (IRS) 1, insulin receptor beta (IR β), and Akt1 were normalized by their corresponding protein contents and PY 20 was normalized by β-actin. ( B ) Hepatic IRS-1-associated PI 3K activity in mice. Liver lysates from basal and insulin-stimulated mice were immunoprecipitated with IRS-1 antibody and protein A agarose. The immune complexes were incubated with reaction buffer containing [γ- 32 P]adenosine 5′-triphosphate, MgCl 2 , MnCl 2 , and phosphatidylinositol for 20 minutes. Autoradiograph was performed after thin-layer chromatography. Notes: Data presented as mean ± standard error of the mean (n = 9/group). * P

Techniques Used: Activity Assay, Mouse Assay, Polyacrylamide Gel Electrophoresis, Immunoprecipitation, Incubation, Autoradiography, Thin Layer Chromatography

57) Product Images from "Targeting TRAF6 E3 ligase activity with a small-molecule inhibitor combats autoimmunity"

Article Title: Targeting TRAF6 E3 ligase activity with a small-molecule inhibitor combats autoimmunity

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.RA118.002649

C25-140 effect on proinflammatory signaling and T-cell activation. A , endogenous TRAF6 auto-ubiquitination ( Ub ) in MEF cells upon IL-1β stimulation is reduced after C25-140 treatment. B , C25-140 impairs IL-1β-induced IκBα phosphorylation. pIκBα levels were densitometrically quantified in relation to β-actin. Error bars , S.D.; n = 3 biological replicates were quantified; unpaired t test (two-tailed); ****, p
Figure Legend Snippet: C25-140 effect on proinflammatory signaling and T-cell activation. A , endogenous TRAF6 auto-ubiquitination ( Ub ) in MEF cells upon IL-1β stimulation is reduced after C25-140 treatment. B , C25-140 impairs IL-1β-induced IκBα phosphorylation. pIκBα levels were densitometrically quantified in relation to β-actin. Error bars , S.D.; n = 3 biological replicates were quantified; unpaired t test (two-tailed); ****, p

Techniques Used: Activation Assay, Two Tailed Test

58) Product Images from "The RNA-binding complex ESCRT-II in Xenopus laevis eggs recognizes purine-rich sequences through its subunit, Vps25"

Article Title: The RNA-binding complex ESCRT-II in Xenopus laevis eggs recognizes purine-rich sequences through its subunit, Vps25

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.RA118.003718

Analysis of ESCRT-II/RNA binding in vitro . A , Coomassie Blue-stained gel of the recombinant Xenopus ( Xen ) and human ( Hu ) ESCRT-II complexes used in the in vitro RNA-binding assays. ΔMBD lacks the membrane-binding domains of human ESCRT-II. B–D , autoradiographs of UV–cross-linked in vitro binding reactions with: B , Xenopus ESCRT-II and 5′-end-labeled total egg RNA; C , Xenopus ESCRT-II and individual 5′-end-labeled in vitro transcribed RNAs that are under-represented in ESCRT-II immunoprecipitations; and D , full-length HuESCRT-II ( FL ) or HuESCRT-IIΔMBD (ΔMBD) and a body-labeled, in vitro transcribed GA-rich CLIP tag (a region of the ctr9 mRNA). B and C , a covalent intermediate of PNK and [γ- 32 P]ATP (used to radiolabel the RNA fragments) is indicated. D , Folch fraction liposomes were included in the binding reactions at the indicated concentrations. A fluorescent Western blotting ( WB ) of the same nitrocellulose membrane shown in the autoradiograph is shown as a loading control. The asterisk represents a nonspecific band. A–D , the expected migrations of the ESCRT-II subunits are indicated. E , quantification of the autoradiograph shown in D and two additional, independent replicates depicting the fraction of RNA bound by each ESCRT-II subunit at the indicated concentrations of Folch fraction liposomes relative to binding with no liposomes present. Error bars are S.E.
Figure Legend Snippet: Analysis of ESCRT-II/RNA binding in vitro . A , Coomassie Blue-stained gel of the recombinant Xenopus ( Xen ) and human ( Hu ) ESCRT-II complexes used in the in vitro RNA-binding assays. ΔMBD lacks the membrane-binding domains of human ESCRT-II. B–D , autoradiographs of UV–cross-linked in vitro binding reactions with: B , Xenopus ESCRT-II and 5′-end-labeled total egg RNA; C , Xenopus ESCRT-II and individual 5′-end-labeled in vitro transcribed RNAs that are under-represented in ESCRT-II immunoprecipitations; and D , full-length HuESCRT-II ( FL ) or HuESCRT-IIΔMBD (ΔMBD) and a body-labeled, in vitro transcribed GA-rich CLIP tag (a region of the ctr9 mRNA). B and C , a covalent intermediate of PNK and [γ- 32 P]ATP (used to radiolabel the RNA fragments) is indicated. D , Folch fraction liposomes were included in the binding reactions at the indicated concentrations. A fluorescent Western blotting ( WB ) of the same nitrocellulose membrane shown in the autoradiograph is shown as a loading control. The asterisk represents a nonspecific band. A–D , the expected migrations of the ESCRT-II subunits are indicated. E , quantification of the autoradiograph shown in D and two additional, independent replicates depicting the fraction of RNA bound by each ESCRT-II subunit at the indicated concentrations of Folch fraction liposomes relative to binding with no liposomes present. Error bars are S.E.

Techniques Used: RNA Binding Assay, In Vitro, Staining, Recombinant, Binding Assay, Labeling, Cross-linking Immunoprecipitation, Western Blot, Autoradiography

59) Product Images from "Atomic-level structure engineering of metal oxides for high-rate oxygen intercalation pseudocapacitance"

Article Title: Atomic-level structure engineering of metal oxides for high-rate oxygen intercalation pseudocapacitance

Journal: Science Advances

doi: 10.1126/sciadv.aau6261

Characterization of Zn x Co 1− x O NRs. ( A ) Typical scanning electron microscopy (SEM) image of Zn x Co 1− x O NR arrays distributed on CFP. ( B ) Transmission electron microscopy (TEM) image of an individual Zn x Co 1− x O NR. ( C ) Elemental mapping of Co, O, and Zn on a single Zn x Co 1− x O NR. ( D ) Atomic-resolution high-angle annular dark-field (HAADF)–scanning TEM (STEM) image of the surface, which is enclosed with {111} nanofacets. ( E ) EELS Co-L 2,3 spectra of Zn x Co 1− x O NR collected at two specific sites (site 1 and site 2) as indicated in (D), where a 0.45-eV peak shift toward the low energy loss direction is evident in site 1 respective to that in site 2. The data presented in this figure refer to Zn 0.04 Co 0.96 O NRs. a.u., arbitrary units.
Figure Legend Snippet: Characterization of Zn x Co 1− x O NRs. ( A ) Typical scanning electron microscopy (SEM) image of Zn x Co 1− x O NR arrays distributed on CFP. ( B ) Transmission electron microscopy (TEM) image of an individual Zn x Co 1− x O NR. ( C ) Elemental mapping of Co, O, and Zn on a single Zn x Co 1− x O NR. ( D ) Atomic-resolution high-angle annular dark-field (HAADF)–scanning TEM (STEM) image of the surface, which is enclosed with {111} nanofacets. ( E ) EELS Co-L 2,3 spectra of Zn x Co 1− x O NR collected at two specific sites (site 1 and site 2) as indicated in (D), where a 0.45-eV peak shift toward the low energy loss direction is evident in site 1 respective to that in site 2. The data presented in this figure refer to Zn 0.04 Co 0.96 O NRs. a.u., arbitrary units.

Techniques Used: Electron Microscopy, Transmission Assay, Transmission Electron Microscopy

60) Product Images from "In Vitro System for Coupling RNAP II Transcription to Primary microRNA Processing and a Three-way System for RNAP II Transcription/Splicing/microRNA Processing"

Article Title: In Vitro System for Coupling RNAP II Transcription to Primary microRNA Processing and a Three-way System for RNAP II Transcription/Splicing/microRNA Processing

Journal: Methods in molecular biology (Clifton, N.J.)

doi: 10.1007/978-1-4939-8624-8_4

Coupled RNAP II transcription/splicing/pri-miRNA processing in vitro. (A) Structure of the CMV-FTZ-let DNA template. The sizes of FTZ pre-mRNA exons, intron, 5’ flanking region, 3’ flanking region, and pre-let-7a are indicated. The thick line indicates pri-miRNA sequences and the thin lines indicate intron sequences. (B) CMV-FTZ-let DNA template was incubated in nuclear extract for 20 min to assemble a pre-initiation complex followed by addition of 32 P-UTP, ATP, and Creatine Phosphate and continued incubation for 10 min. α-Amanitin was added to stop transcription and incubation was continued for the indicated times. Full length transcript, spliced mRNA, exon1, pre-miRNAs and 5’/3’ flanks are indicated. Markers (in base pairs) are indicated and Ori indicates the gel origin. Samples were run for 1 hour. (C) Same as (B), except that samples were run for 2 hours to obtain a better separation of pre-mRNA and 5’/3’ flanks.
Figure Legend Snippet: Coupled RNAP II transcription/splicing/pri-miRNA processing in vitro. (A) Structure of the CMV-FTZ-let DNA template. The sizes of FTZ pre-mRNA exons, intron, 5’ flanking region, 3’ flanking region, and pre-let-7a are indicated. The thick line indicates pri-miRNA sequences and the thin lines indicate intron sequences. (B) CMV-FTZ-let DNA template was incubated in nuclear extract for 20 min to assemble a pre-initiation complex followed by addition of 32 P-UTP, ATP, and Creatine Phosphate and continued incubation for 10 min. α-Amanitin was added to stop transcription and incubation was continued for the indicated times. Full length transcript, spliced mRNA, exon1, pre-miRNAs and 5’/3’ flanks are indicated. Markers (in base pairs) are indicated and Ori indicates the gel origin. Samples were run for 1 hour. (C) Same as (B), except that samples were run for 2 hours to obtain a better separation of pre-mRNA and 5’/3’ flanks.

Techniques Used: In Vitro, Incubation

Coupled RNAP II transcription/pri-miRNA processing in vitro. (A) Structure of the CMV-let-7a DNA template. The sizes of 5’ flanking region, 3’ flanking region, and pre-let-7a are indicated. The thick line indicates the natural pri-miRNA sequences and the thin lines indicate the vector sequences. (B) CMV-let-7a DNA template was incubated in nuclear extract for 20 min to assemble a pre-initiation complex followed by addition of 32 P-UTP, ATP, and Creatine Phosphate and continued incubation for 5 min. α-Amanitin was added to stop transcription and incubation was continued for the indicated times. Pri-miRNAs, pre-miRNAs and 5’/3’ flanks are indicated. The endogenous U6 snRNA and tRNA in the extract are labeled by 32 Markers (in base pairs) are indicated, and Ori indicates the gel origin. (C) A dark exposure of (B).
Figure Legend Snippet: Coupled RNAP II transcription/pri-miRNA processing in vitro. (A) Structure of the CMV-let-7a DNA template. The sizes of 5’ flanking region, 3’ flanking region, and pre-let-7a are indicated. The thick line indicates the natural pri-miRNA sequences and the thin lines indicate the vector sequences. (B) CMV-let-7a DNA template was incubated in nuclear extract for 20 min to assemble a pre-initiation complex followed by addition of 32 P-UTP, ATP, and Creatine Phosphate and continued incubation for 5 min. α-Amanitin was added to stop transcription and incubation was continued for the indicated times. Pri-miRNAs, pre-miRNAs and 5’/3’ flanks are indicated. The endogenous U6 snRNA and tRNA in the extract are labeled by 32 Markers (in base pairs) are indicated, and Ori indicates the gel origin. (C) A dark exposure of (B).

Techniques Used: In Vitro, Plasmid Preparation, Incubation, Labeling

61) Product Images from "Preformed Soluble Chemoreceptor Trimers That Mimic Cellular Assembly States and Activate CheA Autophosphorylation"

Article Title: Preformed Soluble Chemoreceptor Trimers That Mimic Cellular Assembly States and Activate CheA Autophosphorylation

Journal: Biochemistry

doi: 10.1021/bi501570n

CheA autophosphorylation in the presence of CheW and Tar variants or membrane arrays. (A) Phosphor image of a radioisotope PAGE gel of E. coli CheA autophosphorylation with receptor variants with or without CheW. All the receptors increase CheA activity only if CheW is present. E. coli CheA, CheW, and Tar FO or Tar SC (in a 1:1:6 subunit ratio, 2.5 μM CheA) were left to complex at 25 °C for 1 h prior to exposure to [γ- 32 P]ATP for 30 s. Top and bottom gels are shown at different imaging exposures to aid comparisons for the more active species. (B) PAGE gel comparing CheA activity with Tar FO 4Q with and without CheY (40 μM) vs a membrane (Mem.) array comprised of CheA (2.5 μM), CheW (5 μM), and Tsr receptors (3.4 μM). All band intensities are scaled relative to a normalized free CheA control (30 s time point) present on each gel.
Figure Legend Snippet: CheA autophosphorylation in the presence of CheW and Tar variants or membrane arrays. (A) Phosphor image of a radioisotope PAGE gel of E. coli CheA autophosphorylation with receptor variants with or without CheW. All the receptors increase CheA activity only if CheW is present. E. coli CheA, CheW, and Tar FO or Tar SC (in a 1:1:6 subunit ratio, 2.5 μM CheA) were left to complex at 25 °C for 1 h prior to exposure to [γ- 32 P]ATP for 30 s. Top and bottom gels are shown at different imaging exposures to aid comparisons for the more active species. (B) PAGE gel comparing CheA activity with Tar FO 4Q with and without CheY (40 μM) vs a membrane (Mem.) array comprised of CheA (2.5 μM), CheW (5 μM), and Tsr receptors (3.4 μM). All band intensities are scaled relative to a normalized free CheA control (30 s time point) present on each gel.

Techniques Used: Polyacrylamide Gel Electrophoresis, Activity Assay, Imaging

Kinetics of CheA autophosphorylation with Tar variants. E. coli CheA, CheW, and Tar FO 4Q and short or Tar SC (in a 1:1:3 subunit ratio, 1 μM CheA note that the receptor subunit is a single-chain “dimer”) were allowed to complex at 25 °C for 1 h prior to exposure to [γ- 32 P]ATP for the indicated time points. Each data point represents an average over two to four assays. (A) CheA-P formation over time in the presence of CheW and Tar variants. The inset shows CheA-P buildup with Tar FO short compared to CheA:CheW alone out to 30 min. Curves were fit to a first-order kinetic transition (see Experimental Procedures ). (B) Addition of cold ADP to CheA and CheW with or without Tar variants after initial autophosphorylation with [γ- 32 P]ATP for 6 min. (C) Addition of cold ATP to CheA and CheW after incubation with [γ- 32 P]ATP for 6 min. (D) Transfer to CheY in the presence of CheA and CheW with or without Tar FO 4Q and short. Error bars represent the standard error of the mean (SEM) calculated from three independent experiments ( n = 3).
Figure Legend Snippet: Kinetics of CheA autophosphorylation with Tar variants. E. coli CheA, CheW, and Tar FO 4Q and short or Tar SC (in a 1:1:3 subunit ratio, 1 μM CheA note that the receptor subunit is a single-chain “dimer”) were allowed to complex at 25 °C for 1 h prior to exposure to [γ- 32 P]ATP for the indicated time points. Each data point represents an average over two to four assays. (A) CheA-P formation over time in the presence of CheW and Tar variants. The inset shows CheA-P buildup with Tar FO short compared to CheA:CheW alone out to 30 min. Curves were fit to a first-order kinetic transition (see Experimental Procedures ). (B) Addition of cold ADP to CheA and CheW with or without Tar variants after initial autophosphorylation with [γ- 32 P]ATP for 6 min. (C) Addition of cold ATP to CheA and CheW after incubation with [γ- 32 P]ATP for 6 min. (D) Transfer to CheY in the presence of CheA and CheW with or without Tar FO 4Q and short. Error bars represent the standard error of the mean (SEM) calculated from three independent experiments ( n = 3).

Techniques Used: Incubation

62) Product Images from "Interplay between PTB and miR-1285 at the p53 3′UTR modulates the levels of p53 and its isoform Δ40p53α"

Article Title: Interplay between PTB and miR-1285 at the p53 3′UTR modulates the levels of p53 and its isoform Δ40p53α

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkx630

PTB binds specifically to p53 3′UTR. ( A ) α- 32 P-labeled 3′UTR (Lanes 3,4) and α- 32 P-labeled 5′UTR (lanes 5 and 6) were UV cross-linked with increasing concentration of PTB (250 ng, 500 ng), lane 2 is no protein (NP) and lane 1 represents the MW marker in kDa. ( B ) Competition UV crosslinking of PTB and α- 32 P-labeled 3′UTR with 100- and 200-fold molar excess of either unlabeled p53 3′UTR RNA (lanes 4 and 5), unlabeled p53 5′UTR (lanes 6 and 7) or non-specific RNA (Nsp RNA) (lanes 8 and 9) showing the specificity of PTB and 3′UTR binding. Lane 1 represents the MW marker in kDa, lane 2 is no protein (NP) and lane 3 represents binding in the absence of competitor RNA (NC). ( C ) After UV cross-linking of α- 32 P-labeled p53 3′UTR RNA with A549 cell S10 extract, immunoprecipitation was carried out using the respective antibodies, followed by incubation with protein G-Sepharose beads. The beads were either used after saturation with IgG isotype control antibody (lane 2) or anti-PTB antibody (lane 3). Lane 1, no protein (NP); lane 4, 25% of the UV-cross-linked S10 extract (input). Lane 5 is UV-crosslinked recombinant PTB (500 ng) protein. Numbers to the left represent relative mobilities of the molecular mass markers. ( D ) H1299 cells were transfected with Luc-p53 3′UTR construct. After 48 h, the cells were lysed with lysis buffer. RNP complexes were immunoprecipitated with anti- PTB antibody. p53 3′UTR was detected by RT-PCR analysis ( n  = 3).
Figure Legend Snippet: PTB binds specifically to p53 3′UTR. ( A ) α- 32 P-labeled 3′UTR (Lanes 3,4) and α- 32 P-labeled 5′UTR (lanes 5 and 6) were UV cross-linked with increasing concentration of PTB (250 ng, 500 ng), lane 2 is no protein (NP) and lane 1 represents the MW marker in kDa. ( B ) Competition UV crosslinking of PTB and α- 32 P-labeled 3′UTR with 100- and 200-fold molar excess of either unlabeled p53 3′UTR RNA (lanes 4 and 5), unlabeled p53 5′UTR (lanes 6 and 7) or non-specific RNA (Nsp RNA) (lanes 8 and 9) showing the specificity of PTB and 3′UTR binding. Lane 1 represents the MW marker in kDa, lane 2 is no protein (NP) and lane 3 represents binding in the absence of competitor RNA (NC). ( C ) After UV cross-linking of α- 32 P-labeled p53 3′UTR RNA with A549 cell S10 extract, immunoprecipitation was carried out using the respective antibodies, followed by incubation with protein G-Sepharose beads. The beads were either used after saturation with IgG isotype control antibody (lane 2) or anti-PTB antibody (lane 3). Lane 1, no protein (NP); lane 4, 25% of the UV-cross-linked S10 extract (input). Lane 5 is UV-crosslinked recombinant PTB (500 ng) protein. Numbers to the left represent relative mobilities of the molecular mass markers. ( D ) H1299 cells were transfected with Luc-p53 3′UTR construct. After 48 h, the cells were lysed with lysis buffer. RNP complexes were immunoprecipitated with anti- PTB antibody. p53 3′UTR was detected by RT-PCR analysis ( n = 3).

Techniques Used: Labeling, Concentration Assay, Marker, Binding Assay, Immunoprecipitation, Incubation, Recombinant, Transfection, Construct, Lysis, Reverse Transcription Polymerase Chain Reaction

Effect of knockdown of miRNAs on the expression of p53 isoforms and miRNAs interplay with PTB. ( A ) H1299 cells were co-transfected with Luc-3′UTR and anti-miRs for miR-30d/miR-1285/miR-504/miR-181 (10 nM) and Rluc (Renilla luciferase) as transfection control. After 48 h, the cells were lysed and processed for luciferase activity. The graph represents the normalized luciferase activity, i.e. Fluc/Rluc (F/R) ( n  = 3). ( B ) H1299 cells were co-transfected with p53 cDNA construct having 3′UTR (5′UTR + cDNA + 3′UTR) or no 3′UTR (5′UTR + cDNA) and anti-miR-30d/anti-miR-1285 in two concentrations (10 nM and 20 nM). After 48 h, the cells were lysed and processed for immunoblotting. ( C ) Western blot analysis of cell extracts from A549-cells transfected with anti-miRs for miR-1285/miR-30d (30 nM), probed with CM1 after 48 h. Upper panel: p53 and Δ40p53; lower panel: actin. ( D ) Quantitative PCR of  p21  and  14–3-3σ  mRNA levels normalized to Actin in A549 cells transfected with anti-miRs for miR-1285/ miR-30d (30 nM) for 48 h ( n  = 3). ( E ) H1299 cells were co-transfected with Luc-3′UTR and anti-miRs for miR-30d/miR-1285 (20 nM). After 48 h, the cells were lysed and RNP complexes were immunoprecipitated with anti-PTB antibody. RT PCR was performed for checking the levels of 3′UTR associated with PTB. The values for RNA immunoprecipitated with anti PTB antibody in different conditions (no anti-miR, anti-miR-30d and anti-miR-1285) were first normalized with its respective values for RNA immunoprecipitated with IgG control antibody and then the fold change was calculated using the value for 3′UTR pulled down with anti-PTB, without anti-miR treatment as the basal value ( n  = 5). ( F ) H1299 cells were transfected with wild type Fluc -3′UTR (labeled as WT), Fluc -3′UTR mut miR-1285 M1 (3′UTR mutated for miR-1285 one binding site, labeled as M1), Fluc -3′UTR mut miR-1285 M1+M2 (3′UTR mutated for miR-1285 both binding sites, labeled as M1 + M2) and GFP expression from a GFP plasmid vector was used as transfection efficiency control. After 24 h, the cells were lysed and processed for luciferase activity. The graph shows normalized Fluc activity of different constructs ( n  = 3). ( G ) H1299 cells were transfected with either wild type Fluc -3′UTR (labeled as WT) or Fluc -3′UTR mut miR-1285 M1+M2 (3′UTR mutated for miR-1285 both binding sites, labeled as M1 + M2). After 24 h, the cells were lysed and RNP complexes were immunoprecipitated with anti-PTB antibody. RT PCR was performed for checking the levels of 3′UTR associated with PTB. The values for RNA (WT and M1 + M2) immunoprecipitated with anti PTB antibody were first normalized with its respective values for RNA immunoprecipitated with IgG control antibody, and then the fold change was calculated using the value for 3′UTR pulled down with anti-PTB from WT-3′UTR transfected cells as the basal value ( n  = 6). ( H ) H1299 cells were transfected with either wild type Fluc -3′UTR (labeled as WT) or Fluc -3′UTR mut miR-1285 M1+M2 (3′UTR mutated for miR-1285 both binding sites, labeled as M1 + M2). After 24 h, the cells were lysed and RNP complexes were immunoprecipitated with anti-Ago-2 antibody. q-RT PCR was performed for checking the levels of 3′UTR associated with Ago-2. The values for RNA (WT and M1+M2) immunoprecipitated with anti Ago-2 antibody were first normalized with its respective values for RNA immunoprecipitated with IgG control antibody and then the fold change was calculated using the value for 3′UTR pulled down with anti-Ago-2 from WT-3′UTR transfected cells as the basal value ( n  = 6). ( I ) Toe-printing analysis to map the contact points of PTB on p53 3′UTR. p53 3′UTR RNA was incubated in absence (lane 7) and presence (lanes 5 and 6) of increasing concentrations of purified recombinant PTB (200 ng, 400 ng). The RNA in the ribonucleoprotein complexes were reverse transcribed using 3R3 reverse primer (p53 3′UTR region 3 reverse primer) and the resulting cDNAs were resolved in 8% acrylamide–8 M urea PAGE in parallel with a sequencing reaction. The cDNA products terminated at the sites due to protein binding is marked by the arrows. The toe-prints at positions 61 (749T), 55 (755T), 54 (756T), 52 (758T) in the 3′ UTR are indicated. ( J ) Schematic representation of the toe-prints of PTB protein on putative predicted secondary structure of p53 3′UTR R-III (third region) RNA (nucleotides 759–809 in 3′UTR) generated by MFOLD. Nucleotides 748 to 758 bind PTB and are pointed out in red. The curly bracket region shows the miR-1285 binding site (seed sequence). ( K ) α- 32 P-labeled 3′UTR (wild type: WT, mutant: 2a = TT to GG 755–756, 3a = TT to GG 748–749) were UV crosslinked with increasing concentration of PTB (50 ng, 100 ng), lane 1 is no protein (NP). ( L ) H1299 cells were transfected with wild type Luc -3′UTR (labeled as WT), Luc -3′UTR Mut2a and Luc -3′UTR Mut3a (Mutant: 2a = TT to GG 755–756, 3a = TT to GG 748–749) and Rluc was used as transfection control. After 24 h, the cells were lysed and processed for luciferase activity. The graph shows the normalized Fluc activity (F/R) of different constructs ( n  = 6).
Figure Legend Snippet: Effect of knockdown of miRNAs on the expression of p53 isoforms and miRNAs interplay with PTB. ( A ) H1299 cells were co-transfected with Luc-3′UTR and anti-miRs for miR-30d/miR-1285/miR-504/miR-181 (10 nM) and Rluc (Renilla luciferase) as transfection control. After 48 h, the cells were lysed and processed for luciferase activity. The graph represents the normalized luciferase activity, i.e. Fluc/Rluc (F/R) ( n = 3). ( B ) H1299 cells were co-transfected with p53 cDNA construct having 3′UTR (5′UTR + cDNA + 3′UTR) or no 3′UTR (5′UTR + cDNA) and anti-miR-30d/anti-miR-1285 in two concentrations (10 nM and 20 nM). After 48 h, the cells were lysed and processed for immunoblotting. ( C ) Western blot analysis of cell extracts from A549-cells transfected with anti-miRs for miR-1285/miR-30d (30 nM), probed with CM1 after 48 h. Upper panel: p53 and Δ40p53; lower panel: actin. ( D ) Quantitative PCR of p21 and 14–3-3σ mRNA levels normalized to Actin in A549 cells transfected with anti-miRs for miR-1285/ miR-30d (30 nM) for 48 h ( n = 3). ( E ) H1299 cells were co-transfected with Luc-3′UTR and anti-miRs for miR-30d/miR-1285 (20 nM). After 48 h, the cells were lysed and RNP complexes were immunoprecipitated with anti-PTB antibody. RT PCR was performed for checking the levels of 3′UTR associated with PTB. The values for RNA immunoprecipitated with anti PTB antibody in different conditions (no anti-miR, anti-miR-30d and anti-miR-1285) were first normalized with its respective values for RNA immunoprecipitated with IgG control antibody and then the fold change was calculated using the value for 3′UTR pulled down with anti-PTB, without anti-miR treatment as the basal value ( n = 5). ( F ) H1299 cells were transfected with wild type Fluc -3′UTR (labeled as WT), Fluc -3′UTR mut miR-1285 M1 (3′UTR mutated for miR-1285 one binding site, labeled as M1), Fluc -3′UTR mut miR-1285 M1+M2 (3′UTR mutated for miR-1285 both binding sites, labeled as M1 + M2) and GFP expression from a GFP plasmid vector was used as transfection efficiency control. After 24 h, the cells were lysed and processed for luciferase activity. The graph shows normalized Fluc activity of different constructs ( n = 3). ( G ) H1299 cells were transfected with either wild type Fluc -3′UTR (labeled as WT) or Fluc -3′UTR mut miR-1285 M1+M2 (3′UTR mutated for miR-1285 both binding sites, labeled as M1 + M2). After 24 h, the cells were lysed and RNP complexes were immunoprecipitated with anti-PTB antibody. RT PCR was performed for checking the levels of 3′UTR associated with PTB. The values for RNA (WT and M1 + M2) immunoprecipitated with anti PTB antibody were first normalized with its respective values for RNA immunoprecipitated with IgG control antibody, and then the fold change was calculated using the value for 3′UTR pulled down with anti-PTB from WT-3′UTR transfected cells as the basal value ( n = 6). ( H ) H1299 cells were transfected with either wild type Fluc -3′UTR (labeled as WT) or Fluc -3′UTR mut miR-1285 M1+M2 (3′UTR mutated for miR-1285 both binding sites, labeled as M1 + M2). After 24 h, the cells were lysed and RNP complexes were immunoprecipitated with anti-Ago-2 antibody. q-RT PCR was performed for checking the levels of 3′UTR associated with Ago-2. The values for RNA (WT and M1+M2) immunoprecipitated with anti Ago-2 antibody were first normalized with its respective values for RNA immunoprecipitated with IgG control antibody and then the fold change was calculated using the value for 3′UTR pulled down with anti-Ago-2 from WT-3′UTR transfected cells as the basal value ( n = 6). ( I ) Toe-printing analysis to map the contact points of PTB on p53 3′UTR. p53 3′UTR RNA was incubated in absence (lane 7) and presence (lanes 5 and 6) of increasing concentrations of purified recombinant PTB (200 ng, 400 ng). The RNA in the ribonucleoprotein complexes were reverse transcribed using 3R3 reverse primer (p53 3′UTR region 3 reverse primer) and the resulting cDNAs were resolved in 8% acrylamide–8 M urea PAGE in parallel with a sequencing reaction. The cDNA products terminated at the sites due to protein binding is marked by the arrows. The toe-prints at positions 61 (749T), 55 (755T), 54 (756T), 52 (758T) in the 3′ UTR are indicated. ( J ) Schematic representation of the toe-prints of PTB protein on putative predicted secondary structure of p53 3′UTR R-III (third region) RNA (nucleotides 759–809 in 3′UTR) generated by MFOLD. Nucleotides 748 to 758 bind PTB and are pointed out in red. The curly bracket region shows the miR-1285 binding site (seed sequence). ( K ) α- 32 P-labeled 3′UTR (wild type: WT, mutant: 2a = TT to GG 755–756, 3a = TT to GG 748–749) were UV crosslinked with increasing concentration of PTB (50 ng, 100 ng), lane 1 is no protein (NP). ( L ) H1299 cells were transfected with wild type Luc -3′UTR (labeled as WT), Luc -3′UTR Mut2a and Luc -3′UTR Mut3a (Mutant: 2a = TT to GG 755–756, 3a = TT to GG 748–749) and Rluc was used as transfection control. After 24 h, the cells were lysed and processed for luciferase activity. The graph shows the normalized Fluc activity (F/R) of different constructs ( n = 6).

Techniques Used: Expressing, Transfection, Luciferase, Activity Assay, Construct, Western Blot, Real-time Polymerase Chain Reaction, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Labeling, Binding Assay, Plasmid Preparation, Incubation, Purification, Recombinant, Polyacrylamide Gel Electrophoresis, Sequencing, Protein Binding, Generated, Mutagenesis, Concentration Assay

PTB binding regions in p53 3′UTR. ( A ) Mfold-predicted structure of complete p53 3′UTR. ( B ) Different regions of p53 3′UTR used in panel C are shown separately. ( C ) Competition UV crosslinking of α- 32 P-labeled 3′UTR and unlabeled cold RNA of different regions of 3′UTR with PTB. Lane 1: no protein (NP), lane 2: no competition (NC), lane 3 and 4: 100- and 200-fold molar excess of either unlabeled p53 3′UTR region I RNA, lanes 5 and 6: 100- and 200-fold molar excess of either unlabeled p53 3′UTR region II RNA, Lanes 7 and 8: 100- and 200-fold molar excess of either unlabeled p53 3′UTR region III RNA, lanes 9 and 10: 100- and 200-fold molar excess of either unlabeled p53 3′UTR region IV RNA, lanes 11 and 12: 100- and 200-fold molar excess of either unlabeled p53 3′UTRRNA, lanes 13 and 14: non-specific RNA (Nsp RNA).
Figure Legend Snippet: PTB binding regions in p53 3′UTR. ( A ) Mfold-predicted structure of complete p53 3′UTR. ( B ) Different regions of p53 3′UTR used in panel C are shown separately. ( C ) Competition UV crosslinking of α- 32 P-labeled 3′UTR and unlabeled cold RNA of different regions of 3′UTR with PTB. Lane 1: no protein (NP), lane 2: no competition (NC), lane 3 and 4: 100- and 200-fold molar excess of either unlabeled p53 3′UTR region I RNA, lanes 5 and 6: 100- and 200-fold molar excess of either unlabeled p53 3′UTR region II RNA, Lanes 7 and 8: 100- and 200-fold molar excess of either unlabeled p53 3′UTR region III RNA, lanes 9 and 10: 100- and 200-fold molar excess of either unlabeled p53 3′UTR region IV RNA, lanes 11 and 12: 100- and 200-fold molar excess of either unlabeled p53 3′UTRRNA, lanes 13 and 14: non-specific RNA (Nsp RNA).

Techniques Used: Binding Assay, Labeling

SNVs in the p53 3′UTR can affect 3′UTR function. ( A ) A549 cells were transfected with luciferase constructs having either wild type 3′UTR or 3′UTR with the SNVs (SNV 287, SNV 737, SNV 93 and SNV 806). After 12 h and 24 h, the cells were lysed and processed for luciferase activity ( n  = 6). ( B ) H1299 cells were transfected with luciferase constructs having either wild type 3′UTR or 3′UTR with the SNVs (SNV 287, SNV 737, SNV 93 and SNV 806). After 12 h and 24 h, the cells were lysed and processed for luciferase activity ( n  = 6). ( C ) Cytoplamic-S10 extract from A549 cells at three different concentrations) was incubated with α- 32 P UTP labeled p53 3′UTR WT and p53 3′UTR having the SNV 806 (C-T) RNA for UV cross-linking. Protein bands in the box show that PTB binding in both S10 and recombinant protein is more with p53 3′UTR having the SNV 806 (C-T) as compared to WT 3′UTR.
Figure Legend Snippet: SNVs in the p53 3′UTR can affect 3′UTR function. ( A ) A549 cells were transfected with luciferase constructs having either wild type 3′UTR or 3′UTR with the SNVs (SNV 287, SNV 737, SNV 93 and SNV 806). After 12 h and 24 h, the cells were lysed and processed for luciferase activity ( n = 6). ( B ) H1299 cells were transfected with luciferase constructs having either wild type 3′UTR or 3′UTR with the SNVs (SNV 287, SNV 737, SNV 93 and SNV 806). After 12 h and 24 h, the cells were lysed and processed for luciferase activity ( n = 6). ( C ) Cytoplamic-S10 extract from A549 cells at three different concentrations) was incubated with α- 32 P UTP labeled p53 3′UTR WT and p53 3′UTR having the SNV 806 (C-T) RNA for UV cross-linking. Protein bands in the box show that PTB binding in both S10 and recombinant protein is more with p53 3′UTR having the SNV 806 (C-T) as compared to WT 3′UTR.

Techniques Used: Transfection, Luciferase, Construct, Activity Assay, Incubation, Labeling, Binding Assay, Recombinant

63) Product Images from "Active site–adjacent phosphorylation at Tyr-397 by c-Abl kinase inactivates caspase-9"

Article Title: Active site–adjacent phosphorylation at Tyr-397 by c-Abl kinase inactivates caspase-9

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M117.811976

c-Abl phosphorylates casp-9 in vitro at the small subunit. A and C , recombinant c-Abl constructs used to phosphorylate casp-9 in vitro . The construct c-Abl KD comprises only the kinase domain, whereas the c-Abl 3D construct contains the SH3-SH2 regulatory/binding domains as well as the kinase domain. B and D , casp-9 catalytic site–inactivated variant C287A (full-length) and WT (cleaved) were subjected to in vitro phosphorylation by c-Abl KD or 3D in the presence of ATP + [γ- 32 P]ATP for 2 h. c-Abl undergoes autophosphorylation/autoactivation upon treatment with ATP. Both forms of c-Abl phosphorylated casp-9 in the zymogen (C287A) and cleaved (WT) forms. No phosphorylation in the CARD+Large region (Tyr-153 site) was detected, but phosphorylation in the small subunit was clearly visible, as shown in the autoradiograph labeled here and in the succeeding figures as 32 P .
Figure Legend Snippet: c-Abl phosphorylates casp-9 in vitro at the small subunit. A and C , recombinant c-Abl constructs used to phosphorylate casp-9 in vitro . The construct c-Abl KD comprises only the kinase domain, whereas the c-Abl 3D construct contains the SH3-SH2 regulatory/binding domains as well as the kinase domain. B and D , casp-9 catalytic site–inactivated variant C287A (full-length) and WT (cleaved) were subjected to in vitro phosphorylation by c-Abl KD or 3D in the presence of ATP + [γ- 32 P]ATP for 2 h. c-Abl undergoes autophosphorylation/autoactivation upon treatment with ATP. Both forms of c-Abl phosphorylated casp-9 in the zymogen (C287A) and cleaved (WT) forms. No phosphorylation in the CARD+Large region (Tyr-153 site) was detected, but phosphorylation in the small subunit was clearly visible, as shown in the autoradiograph labeled here and in the succeeding figures as 32 P .

Techniques Used: In Vitro, Recombinant, Construct, Binding Assay, Variant Assay, Autoradiography, Labeling

64) Product Images from "Restricting Conformational Flexibility of the Switch II Region Creates a Dominant-Inhibitory Phenotype in Obg GTPase Nog1 ▿"

Article Title: Restricting Conformational Flexibility of the Switch II Region Creates a Dominant-Inhibitory Phenotype in Obg GTPase Nog1 ▿

Journal:

doi: 10.1128/MCB.01161-07

Guanine nucleotide binding by Nog1 and Nog1G224A. (A) Purified NG fragments of the wild-type (WT) Nog1 and G224A mutant were UV cross-linked with [α- 32 P]GTP in the absence (−) or presence of a 500-fold excess of unlabeled nucleotides as
Figure Legend Snippet: Guanine nucleotide binding by Nog1 and Nog1G224A. (A) Purified NG fragments of the wild-type (WT) Nog1 and G224A mutant were UV cross-linked with [α- 32 P]GTP in the absence (−) or presence of a 500-fold excess of unlabeled nucleotides as

Techniques Used: Binding Assay, Purification, Mutagenesis

65) Product Images from "Extracellular Signal-Regulated Kinase Promotes Rho-Dependent Focal Adhesion Formation by Suppressing p190A RhoGAP ▿"

Article Title: Extracellular Signal-Regulated Kinase Promotes Rho-Dependent Focal Adhesion Formation by Suppressing p190A RhoGAP ▿

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.01178-09

p190A RhoGAP is an ERK substrate. (A) Four potential ERK phosphorylation motifs are highly conserved in the p190A C terminus. Vertebrate p190A sequences are aligned to show potential ERK phosphorylation (PXSP and PXTP) motifs. (B) Collation of putative phosphorylation and docking sites identified either manually or through computational methods (ScanSite). (C and D) p190A is phosphorylated on one or more putative C-terminal ERK sites in vitro and in vivo . FLAG-tagged wild-type (WT) or p190A-4A mutant (4A; where the four putative ERK sites shown in panel A have been replaced with alanines) was immunoprecipitated, pretreated with alkaline phosphatase, and then phosphorylated in vitro with recombinant active ERK in the presence of [γ- 32 P]ATP (C). Alternatively, REF52 cells transfected with FLAG-p190A, FLAG-p190A-4A, or empty vector were metabolically labeled with 32 P i and FLAG-p190A forms were recovered by immunoprecipitation (D). (E) Tryptic digests of in vitro - and in vivo -labeled p190A were resolved by electrophoresis (with low-molecular-mass markers) and transferred to membrane. The positions of the dye front and ∼6.5-, 10-, and 15-kDa markers are indicated. 32 P denotes phosphorimages, and CB in panel C represents a Coomassie blue-stained image of the same blot.
Figure Legend Snippet: p190A RhoGAP is an ERK substrate. (A) Four potential ERK phosphorylation motifs are highly conserved in the p190A C terminus. Vertebrate p190A sequences are aligned to show potential ERK phosphorylation (PXSP and PXTP) motifs. (B) Collation of putative phosphorylation and docking sites identified either manually or through computational methods (ScanSite). (C and D) p190A is phosphorylated on one or more putative C-terminal ERK sites in vitro and in vivo . FLAG-tagged wild-type (WT) or p190A-4A mutant (4A; where the four putative ERK sites shown in panel A have been replaced with alanines) was immunoprecipitated, pretreated with alkaline phosphatase, and then phosphorylated in vitro with recombinant active ERK in the presence of [γ- 32 P]ATP (C). Alternatively, REF52 cells transfected with FLAG-p190A, FLAG-p190A-4A, or empty vector were metabolically labeled with 32 P i and FLAG-p190A forms were recovered by immunoprecipitation (D). (E) Tryptic digests of in vitro - and in vivo -labeled p190A were resolved by electrophoresis (with low-molecular-mass markers) and transferred to membrane. The positions of the dye front and ∼6.5-, 10-, and 15-kDa markers are indicated. 32 P denotes phosphorimages, and CB in panel C represents a Coomassie blue-stained image of the same blot.

Techniques Used: In Vitro, In Vivo, Mutagenesis, Immunoprecipitation, Recombinant, Transfection, Plasmid Preparation, Metabolic Labelling, Labeling, Electrophoresis, Staining

66) Product Images from "Development of a Staphylococcus aureus reporter strain with click beetle red luciferase for enhanced in vivo imaging of experimental bacteremia and mixed infections"

Article Title: Development of a Staphylococcus aureus reporter strain with click beetle red luciferase for enhanced in vivo imaging of experimental bacteremia and mixed infections

Journal: Scientific Reports

doi: 10.1038/s41598-019-52982-0

Dual in vivo BLI monitoring of a mixed S. aureus and P. aeruginosa in vivo wound infection mouse model. A excisional wound mixed infection model by performing a 6-mm punch biopsy on the backs of mice and the wound beds were inoculated with S. aureus AH4775 ( luc ) (2 × 10 6 CFU) and P. aeruginosa Xen41 ( lux ) (2 × 10 5 CFU) (n = 5 mice). The mixed wound infection was followed for 7 days and CFU from the wounds were harvested, plated, cultured and enumerated. To distinguish between the in vivo BLI signals of AH4775 ( luc ) versus Xen41 ( lux ), the respective 670 nm and 520 nm emission filters (IVIS Lumina III) were used because there was no overlap between the luc and lux bioluminescent signals. ( A ) Representative images of the in vivo BLI signals from AH4775 ( luc ) and Xen41 ( lux ) from the wounds. ( B ) Mean in vivo BLI signals of AH4775 ( luc ) and Xen41 ( lux ) quantified as total flux (photons/s) ± SEM. ( C ) Representative in vivo BLI of bacterial culture plates possessing CFU of AH4775 ( luc ) and Xen41 ( lux ) using the 670 nm and 520 nm emission filters, respectively. ( D ) Mean CFU of AH4775 ( luc ) and Xen41 ( lux ), with horizontal bars = geometric mean. * P
Figure Legend Snippet: Dual in vivo BLI monitoring of a mixed S. aureus and P. aeruginosa in vivo wound infection mouse model. A excisional wound mixed infection model by performing a 6-mm punch biopsy on the backs of mice and the wound beds were inoculated with S. aureus AH4775 ( luc ) (2 × 10 6 CFU) and P. aeruginosa Xen41 ( lux ) (2 × 10 5 CFU) (n = 5 mice). The mixed wound infection was followed for 7 days and CFU from the wounds were harvested, plated, cultured and enumerated. To distinguish between the in vivo BLI signals of AH4775 ( luc ) versus Xen41 ( lux ), the respective 670 nm and 520 nm emission filters (IVIS Lumina III) were used because there was no overlap between the luc and lux bioluminescent signals. ( A ) Representative images of the in vivo BLI signals from AH4775 ( luc ) and Xen41 ( lux ) from the wounds. ( B ) Mean in vivo BLI signals of AH4775 ( luc ) and Xen41 ( lux ) quantified as total flux (photons/s) ± SEM. ( C ) Representative in vivo BLI of bacterial culture plates possessing CFU of AH4775 ( luc ) and Xen41 ( lux ) using the 670 nm and 520 nm emission filters, respectively. ( D ) Mean CFU of AH4775 ( luc ) and Xen41 ( lux ), with horizontal bars = geometric mean. * P

Techniques Used: In Vivo, Infection, Mouse Assay, Cell Culture

67) Product Images from "Role of Mitogen-Activated Protein Kinase Activation in Injured and Intact Primary Afferent Neurons for Mechanical and Heat Hypersensitivity after Spinal Nerve Ligation"

Article Title: Role of Mitogen-Activated Protein Kinase Activation in Injured and Intact Primary Afferent Neurons for Mechanical and Heat Hypersensitivity after Spinal Nerve Ligation

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.3388-04.2004

A-F , Effects of intrathecal infusion of anti-NGF on the L5 SNL-induced heat hypersensitivity and increase in p-p38, BDNF, and TRPV1 expression in the L4 DRG. A , Bar graph showing the difference scores to the radiant heat stimuli. Data from the vehicle
Figure Legend Snippet: A-F , Effects of intrathecal infusion of anti-NGF on the L5 SNL-induced heat hypersensitivity and increase in p-p38, BDNF, and TRPV1 expression in the L4 DRG. A , Bar graph showing the difference scores to the radiant heat stimuli. Data from the vehicle

Techniques Used: Expressing

In the injured L5 DRG, L5 SNL induces an increase in p-ERK, p-p38, and p-JNK expression in different populations of DRG neurons and satellite glial cells. Photomicrographs show p-ERK ( A, B ), p-p38 ( D, E ), and p-JNK ( G, H ) immunoreactivity in the ipsilateral
Figure Legend Snippet: In the injured L5 DRG, L5 SNL induces an increase in p-ERK, p-p38, and p-JNK expression in different populations of DRG neurons and satellite glial cells. Photomicrographs show p-ERK ( A, B ), p-p38 ( D, E ), and p-JNK ( G, H ) immunoreactivity in the ipsilateral

Techniques Used: Expressing

p38 activation regulates BDNF and TRPV1 expression in the uninjured L4 DRG
Figure Legend Snippet: p38 activation regulates BDNF and TRPV1 expression in the uninjured L4 DRG

Techniques Used: Activation Assay, Expressing

In the uninjured L4 DRG, L5 SNL induced a sustained increase in p-p38, but not p-ERK or p-JNK, mainly in small and medium-sized neurons. A, B , Photomicrographs showing p-p38 immunoreactivity in the ipsilateral ( A ) and contralateral ( B ) L4 DRG at 7 d after
Figure Legend Snippet: In the uninjured L4 DRG, L5 SNL induced a sustained increase in p-p38, but not p-ERK or p-JNK, mainly in small and medium-sized neurons. A, B , Photomicrographs showing p-p38 immunoreactivity in the ipsilateral ( A ) and contralateral ( B ) L4 DRG at 7 d after

Techniques Used:

Effects of the p38 inhibitor SB203580 delivered intrathecally on L5 SNL-induced BDNF and TRPV1 expression in the uninjured L4 DRG. A, B , Photomicrographs showing the expression of BDNF mRNA ( A, B ) and TRPV1 mRNA ( D, E ) in the L4 DRG in the vehicle and
Figure Legend Snippet: Effects of the p38 inhibitor SB203580 delivered intrathecally on L5 SNL-induced BDNF and TRPV1 expression in the uninjured L4 DRG. A, B , Photomicrographs showing the expression of BDNF mRNA ( A, B ) and TRPV1 mRNA ( D, E ) in the L4 DRG in the vehicle and

Techniques Used: Expressing

p38 is activated in small, presumably nociceptive DRG neurons, but not satellite glial cells in the uninjured L4 DRG after L5 SNL. Immunohistochemical colocalization of green reaction product for p-p38 ( A, D, G ) and red product for NF200 ( B ), GFAP ( E
Figure Legend Snippet: p38 is activated in small, presumably nociceptive DRG neurons, but not satellite glial cells in the uninjured L4 DRG after L5 SNL. Immunohistochemical colocalization of green reaction product for p-p38 ( A, D, G ) and red product for NF200 ( B ), GFAP ( E

Techniques Used: Immunohistochemistry

NGF, p38 activation, and BDNF and TRPV1 expression in the uninjured L4 DRG
Figure Legend Snippet: NGF, p38 activation, and BDNF and TRPV1 expression in the uninjured L4 DRG

Techniques Used: Activation Assay, Expressing

68) Product Images from "Analgesic Properties of Opioid/NK1 Multitarget Ligands with Distinct in Vitro Profiles in Naive and Chronic Constriction Injury Mice"

Article Title: Analgesic Properties of Opioid/NK1 Multitarget Ligands with Distinct in Vitro Profiles in Naive and Chronic Constriction Injury Mice

Journal: ACS chemical neuroscience

doi: 10.1021/acschemneuro.7b00226

The mRNA (left panel) and protein (right panel) level of MOP, DOP and NK1 receptors and substance P in lumbar spinal cord of CCI-exposed mice, on the 7 th  day after surgical procedure. The data are presented as the mean ± SEM. Intergroup differences
Figure Legend Snippet: The mRNA (left panel) and protein (right panel) level of MOP, DOP and NK1 receptors and substance P in lumbar spinal cord of CCI-exposed mice, on the 7 th day after surgical procedure. The data are presented as the mean ± SEM. Intergroup differences

Techniques Used: Mouse Assay

69) Product Images from "Functional Characterization of Monomeric GTPase Rab1 in the Secretory Pathway of Leishmania"

Article Title: Functional Characterization of Monomeric GTPase Rab1 in the Secretory Pathway of Leishmania

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M115.670018

Characterization of LdRab1 and its mutants. A , GTP binding of purified LdRab1:WT and its mutants was detected using an [α- 32 P]GTP overlay assay. LdRab5:WT and GST proteins were used as control. B , GTPase activity of LdRab1 and its mutants was determined as described under “Experimental Procedures.” C , to determine the levels of overexpression of LdRab1:WT and its mutants as GFP fusion proteins in Leishmania , cell lysates were analyzed by Western blotting using anti-GFP antibody. Untransfected Leishmania was used as control. D , to determine the levels of overexpression of LdRab1:WT and its mutants as GFP fusion proteins in Leishmania , cell lysates were analyzed by Western blotting using anti-LdRab1 antibody. Untransfected Leishmania was used as control. E , the same membrane was exposed for a longer duration to detect endogenous Rab1. F , to determine the localization of Rab1:WT and its mutants in Leishmania , cells were transfected with indicated constructs to overexpress the respective protein in Leishmania as GFP fusion protein. Cells were visualized in a LSM 510 Meta confocal microscope. Green , localization of the indicated LdRab1; blue , nucleus ( Nu ). Results are representative of three independent preparations.
Figure Legend Snippet: Characterization of LdRab1 and its mutants. A , GTP binding of purified LdRab1:WT and its mutants was detected using an [α- 32 P]GTP overlay assay. LdRab5:WT and GST proteins were used as control. B , GTPase activity of LdRab1 and its mutants was determined as described under “Experimental Procedures.” C , to determine the levels of overexpression of LdRab1:WT and its mutants as GFP fusion proteins in Leishmania , cell lysates were analyzed by Western blotting using anti-GFP antibody. Untransfected Leishmania was used as control. D , to determine the levels of overexpression of LdRab1:WT and its mutants as GFP fusion proteins in Leishmania , cell lysates were analyzed by Western blotting using anti-LdRab1 antibody. Untransfected Leishmania was used as control. E , the same membrane was exposed for a longer duration to detect endogenous Rab1. F , to determine the localization of Rab1:WT and its mutants in Leishmania , cells were transfected with indicated constructs to overexpress the respective protein in Leishmania as GFP fusion protein. Cells were visualized in a LSM 510 Meta confocal microscope. Green , localization of the indicated LdRab1; blue , nucleus ( Nu ). Results are representative of three independent preparations.

Techniques Used: Binding Assay, Purification, GTP Overlay Assay, Activity Assay, Over Expression, Western Blot, Transfection, Construct, Microscopy

70) Product Images from "Mutating the Conserved Q-loop Glutamine 1291 Selectively Disrupts Adenylate Kinase-dependent Channel Gating of the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and Reduces Channel Function in Primary Human Airway Epithelia *"

Article Title: Mutating the Conserved Q-loop Glutamine 1291 Selectively Disrupts Adenylate Kinase-dependent Channel Gating of the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and Reduces Channel Function in Primary Human Airway Epithelia *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.611616

Adenylate kinase activity of wild-type ( Q1291 ) and Q1291F CFTR. A , left , autoradiograph of immunoprecipitated wild-type and Q1291F CFTR fractionated on a 6% SDS-polyacrylamide gel. Membranes containing 50 μg of protein from either wild-type or Q1291F CFTR-expressing HeLa cells were used in each reaction. Membranes were incubated together with 50 μ m non-radioactive 2-N 3 -AMP and 30 μCi of [γ- 32 P]GTP (6000 Ci/mmol) for 5 min at 37 °C followed by UV irradiation for 30 s, as indicated below each lane. Right , Western blot probed with CFTR antibody 13-4 of 50 μg of the membrane protein preparations used in the left panel. Letters label highly glycosylated ( C ) and core-glycosylated ( B ) CFTR. B , quantitative data. Radioactivity incorporated into CFTR band was quantified by digital autoradiography and normalized to the amount of CFTR protein measured by Western blot as described in the legend to Fig. 10 B . *, p
Figure Legend Snippet: Adenylate kinase activity of wild-type ( Q1291 ) and Q1291F CFTR. A , left , autoradiograph of immunoprecipitated wild-type and Q1291F CFTR fractionated on a 6% SDS-polyacrylamide gel. Membranes containing 50 μg of protein from either wild-type or Q1291F CFTR-expressing HeLa cells were used in each reaction. Membranes were incubated together with 50 μ m non-radioactive 2-N 3 -AMP and 30 μCi of [γ- 32 P]GTP (6000 Ci/mmol) for 5 min at 37 °C followed by UV irradiation for 30 s, as indicated below each lane. Right , Western blot probed with CFTR antibody 13-4 of 50 μg of the membrane protein preparations used in the left panel. Letters label highly glycosylated ( C ) and core-glycosylated ( B ) CFTR. B , quantitative data. Radioactivity incorporated into CFTR band was quantified by digital autoradiography and normalized to the amount of CFTR protein measured by Western blot as described in the legend to Fig. 10 B . *, p

Techniques Used: Activity Assay, Autoradiography, Immunoprecipitation, Expressing, Incubation, Irradiation, Western Blot, Radioactivity

71) Product Images from "Structure and mechanism of a Hypr GGDEF enzyme that activates cGAMP signaling to control extracellular metal respiration"

Article Title: Structure and mechanism of a Hypr GGDEF enzyme that activates cGAMP signaling to control extracellular metal respiration

Journal: eLife

doi: 10.7554/eLife.43959

Unlike DncV and cGAS, GacA uses either substrate in the first bond-forming step. ( A ) Reaction pathways to form cyclic GMP-AMP by different dinucleotide cyclases, DncV from Vibrio cholerae , cGAS from mammalian cells, and GacA from Geobacter sulfurreducens . ( B ) Cellulose TLC analysis of radiolabeled products from enzymatic reactions with MBP-tagged GacA R393A (I-site mutant) with NTP substrates and nonhydrolyzable analogues. Trace amounts of α- 32 P-labeled ATP or α- 32 P-labeled GTP was doped in the reactions. Reactions were quenched with alkaline phosphatase to digest unreacted nucleotides, resulting in production of inorganic phosphate (P i ).
Figure Legend Snippet: Unlike DncV and cGAS, GacA uses either substrate in the first bond-forming step. ( A ) Reaction pathways to form cyclic GMP-AMP by different dinucleotide cyclases, DncV from Vibrio cholerae , cGAS from mammalian cells, and GacA from Geobacter sulfurreducens . ( B ) Cellulose TLC analysis of radiolabeled products from enzymatic reactions with MBP-tagged GacA R393A (I-site mutant) with NTP substrates and nonhydrolyzable analogues. Trace amounts of α- 32 P-labeled ATP or α- 32 P-labeled GTP was doped in the reactions. Reactions were quenched with alkaline phosphatase to digest unreacted nucleotides, resulting in production of inorganic phosphate (P i ).

Techniques Used: Thin Layer Chromatography, Mutagenesis, Labeling

72) Product Images from "Characterization and Expression of HmuR, a TonB-Dependent Hemoglobin Receptor of Porphyromonas gingivalis"

Article Title: Characterization and Expression of HmuR, a TonB-Dependent Hemoglobin Receptor of Porphyromonas gingivalis

Journal: Journal of Bacteriology

doi:

Growth of P. gingivalis A7436 (A) and WS1 with hemin, hemoglobin, and ferric chloride. Cultures were initially starved in Schaedler broth supplemented with 150 μM dipyridyl for 24 h. This was used to inoculate Schaedler broth alone (SB), Schaedler broth plus 150 μM dipyridyl (dip), Schaedler broth plus 150 μM dipyridyl plus 1.5 μM hemin (Hemin), Schaedler broth plus 150 μM dipyridyl plus 1.5 μM hemoglobin (Hb), or Schaedler broth plus 150 μM dipyridyl plus 100 μM ferric chloride (Fc). The results are representative of two experiments.
Figure Legend Snippet: Growth of P. gingivalis A7436 (A) and WS1 with hemin, hemoglobin, and ferric chloride. Cultures were initially starved in Schaedler broth supplemented with 150 μM dipyridyl for 24 h. This was used to inoculate Schaedler broth alone (SB), Schaedler broth plus 150 μM dipyridyl (dip), Schaedler broth plus 150 μM dipyridyl plus 1.5 μM hemin (Hemin), Schaedler broth plus 150 μM dipyridyl plus 1.5 μM hemoglobin (Hb), or Schaedler broth plus 150 μM dipyridyl plus 100 μM ferric chloride (Fc). The results are representative of two experiments.

Techniques Used:

73) Product Images from "Enhanced uptake of potassium or glycine betaine or export of cyclic-di-AMP restores osmoresistance in a high cyclic-di-AMP Lactococcus lactis mutant"

Article Title: Enhanced uptake of potassium or glycine betaine or export of cyclic-di-AMP restores osmoresistance in a high cyclic-di-AMP Lactococcus lactis mutant

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1007574

C-di-AMP levels in non-growing energised cells respond rapidly to environmental osmolarity changes. (A) Schematic diagram showing the steps in the non-growing cell suspension assay. Cells are suspended in a low osmolarity buffer causing water influx. Glucose is added to energise the cells at time 0 to provide ATP for c-di-AMP synthesis. After 10 mins, either water or NaCl/KCl is added causing further cellular hydration or cellular dehydration, respectively. (B) C-di-AMP levels in Lc . lactis WT suspended in different solvents/solutions: ACN-MeOH (acetonitrile:methanol) or buffer (1/10 KPM) with and without glucose and deoxyglucose. (C) C-di-AMP levels in Lc . lactis WT cells in 1/10 KPM buffer + glucose. After the 10 min (arrow), cells were treated with water, 0.1M NaCl, 0.3M NaCl, 0.1 KCl and 0.3M KCl and c-di-AMP levels were measured at 20 mins. The same experiment was carried out for cells of Lb . plantarum (D) , L . monocytogenes (E) , S . aureus (F) , and Lc . lactis Δ gdpP/cdaA T273fs (H) with the key for treatments the same as that shown in (C) . Non-ionic treatments (sucrose and sorbitol) were also performed using L . monocytogenes (G) . Mean ± SEM levels of c-di-AMP were measured from three independent cell suspensions. P
Figure Legend Snippet: C-di-AMP levels in non-growing energised cells respond rapidly to environmental osmolarity changes. (A) Schematic diagram showing the steps in the non-growing cell suspension assay. Cells are suspended in a low osmolarity buffer causing water influx. Glucose is added to energise the cells at time 0 to provide ATP for c-di-AMP synthesis. After 10 mins, either water or NaCl/KCl is added causing further cellular hydration or cellular dehydration, respectively. (B) C-di-AMP levels in Lc . lactis WT suspended in different solvents/solutions: ACN-MeOH (acetonitrile:methanol) or buffer (1/10 KPM) with and without glucose and deoxyglucose. (C) C-di-AMP levels in Lc . lactis WT cells in 1/10 KPM buffer + glucose. After the 10 min (arrow), cells were treated with water, 0.1M NaCl, 0.3M NaCl, 0.1 KCl and 0.3M KCl and c-di-AMP levels were measured at 20 mins. The same experiment was carried out for cells of Lb . plantarum (D) , L . monocytogenes (E) , S . aureus (F) , and Lc . lactis Δ gdpP/cdaA T273fs (H) with the key for treatments the same as that shown in (C) . Non-ionic treatments (sucrose and sorbitol) were also performed using L . monocytogenes (G) . Mean ± SEM levels of c-di-AMP were measured from three independent cell suspensions. P

Techniques Used:

74) Product Images from "Biochemical and Molecular Characterization of Barley Plastidial ADP-Glucose Transporter (HvBT1)"

Article Title: Biochemical and Molecular Characterization of Barley Plastidial ADP-Glucose Transporter (HvBT1)

Journal: PLoS ONE

doi: 10.1371/journal.pone.0098524

Transport activity of HvBT1 in intact E. coli cells. Escherichia coli C43 cells harboring the recombinant plasmid and the empty one as a control were incubated with different concentrations of [α- 32 P] ADP-Glc. The cells were incubated at 30°C for 10 min. The control values have been subtracted. The data are the mean ± SE of three independent experiments, each with three replicates. A : K m value of ADP-glucose is 614.5±33.24 µM and V max of 254.14 ±19.45 nmol of ADP-Glc mg of protein −1 h −1 . B : K m and V max values of ADP is 334.7±39.3 µM and of 47.07±3.51 nmol of ADP-Glc mg of protein −1 h −1 , respectively.
Figure Legend Snippet: Transport activity of HvBT1 in intact E. coli cells. Escherichia coli C43 cells harboring the recombinant plasmid and the empty one as a control were incubated with different concentrations of [α- 32 P] ADP-Glc. The cells were incubated at 30°C for 10 min. The control values have been subtracted. The data are the mean ± SE of three independent experiments, each with three replicates. A : K m value of ADP-glucose is 614.5±33.24 µM and V max of 254.14 ±19.45 nmol of ADP-Glc mg of protein −1 h −1 . B : K m and V max values of ADP is 334.7±39.3 µM and of 47.07±3.51 nmol of ADP-Glc mg of protein −1 h −1 , respectively.

Techniques Used: Activity Assay, Recombinant, Plasmid Preparation, Incubation, Gas Chromatography

Exchange of the intracellular radiolabeled substrates. A : E. coli cells harboring the vector containing HvBT1 and the control vector were incubated with 1 µM [α- 32 P] ADP-Glc at 30°C for 5 min. The assay buffer was diluted with non-labeled ATP, ADP, AMP, and ADP-Glc for indicated time points. The cells were filtered and washed under vacuum, and then measured for radioactivity. The data presented here are the mean ± SE of three independent experiments, each with three replicates. B : the procedures for ADP efflux assay was performed as described for ADP-Glc in (A) with ADP and ADP-Glc dilutions. C : E. coli C43 cells harboring the vector containing HvBT1 and the control vector were preloaded with nucleotides at a final concentration of 1 mM, and then the cells were incubated at 30°C for 5 min. The cells were centrifuged and re-suspended in potassium phosphate buffer (50 mM, pH 7.2) with [α- 32 P] ADP-Glc at concentration of 100 µM at 30°C for 8 min. The data presented are the mean ± SE of three independent experiments, each with three replicates.
Figure Legend Snippet: Exchange of the intracellular radiolabeled substrates. A : E. coli cells harboring the vector containing HvBT1 and the control vector were incubated with 1 µM [α- 32 P] ADP-Glc at 30°C for 5 min. The assay buffer was diluted with non-labeled ATP, ADP, AMP, and ADP-Glc for indicated time points. The cells were filtered and washed under vacuum, and then measured for radioactivity. The data presented here are the mean ± SE of three independent experiments, each with three replicates. B : the procedures for ADP efflux assay was performed as described for ADP-Glc in (A) with ADP and ADP-Glc dilutions. C : E. coli C43 cells harboring the vector containing HvBT1 and the control vector were preloaded with nucleotides at a final concentration of 1 mM, and then the cells were incubated at 30°C for 5 min. The cells were centrifuged and re-suspended in potassium phosphate buffer (50 mM, pH 7.2) with [α- 32 P] ADP-Glc at concentration of 100 µM at 30°C for 8 min. The data presented are the mean ± SE of three independent experiments, each with three replicates.

Techniques Used: Plasmid Preparation, Incubation, Gas Chromatography, Labeling, Radioactivity, Concentration Assay

75) Product Images from "In Vitro Epsilon RNA-Dependent Protein Priming Activity of Human Hepatitis B Virus Polymerase"

Article Title: In Vitro Epsilon RNA-Dependent Protein Priming Activity of Human Hepatitis B Virus Polymerase

Journal: Journal of Virology

doi: 10.1128/JVI.07137-11

Analysis of DP protein priming products by Tdp2 cleavage of the phosphotyrosyl bond between DNA and protein. Purified DP bound to M2 antibody affinity beads was assayed for protein priming. Free nucleotides were then removed with extensive washing, and priming products were mock treated (−) or treated with Tdp2 (+) to cleave the phosphotyrosyl-DNA linkages between DP and the linked nucleotides or DNA oligomers. The supernatant, which contained the released nucleotides/DNA, was collected and resolved on a urea–20% polyacrylamide gel (B). The beads, which contained the primed DP, were processed for SDS-PAGE to visualize the labeled DP (A). Radiolabeled proteins and nucleotides/DNA were detected by autoradiography. Priming was done in the presence of TMgNK buffer and [α- 32 P]dGTP (A, lanes 1 and 2; B, lanes 5 and 6) or TMnNK buffer and [α- 32 P]dGTP plus the unlabeled dCTP, TTP, and dATP (A, lanes 3 and 4; B, lanes 7 and 8). (C) [α- 32 P]dGTP stock was mock (lane 4) or apyrase treated (lane 5). The DP priming product obtained in TMgNK buffer and [α- 32 P]dGTP was either mock treated (lane 2) or Tdp2 treated (lane 3), which released dGMP from the DP-dGMP phosphotyrosyl linkage. Samples were resolved on a urea–20% polyacrylamide gel. The positions of  32 P-labeled 10-nucleotide marker (Invitrogen) (B) and DNA oligomers (dTG, dTGA, and dTGAA in panels B and C) are indicated, as are the positions of dGTP and dGMP. (D) HPLC analysis of dGTP and dGMP. (Panel 1) UV ( A 260 ) detection showing retention times of unlabeled dGMP and dGTP. (Panel 2) Detection of  32 P radioactivity from mock-treated DP priming products (−Tdp2), showing the absence of dGMP and the presence of residual dGTP substrate input. (Panel 3) Detection of  32 P radioactivity from Tdp2-treated DP priming products (+Tdp2), showing the presence of dGMP released by Tdp2 from DP and again some residual dGTP substrate input. The positions of dGMP and dGTP are indicated.
Figure Legend Snippet: Analysis of DP protein priming products by Tdp2 cleavage of the phosphotyrosyl bond between DNA and protein. Purified DP bound to M2 antibody affinity beads was assayed for protein priming. Free nucleotides were then removed with extensive washing, and priming products were mock treated (−) or treated with Tdp2 (+) to cleave the phosphotyrosyl-DNA linkages between DP and the linked nucleotides or DNA oligomers. The supernatant, which contained the released nucleotides/DNA, was collected and resolved on a urea–20% polyacrylamide gel (B). The beads, which contained the primed DP, were processed for SDS-PAGE to visualize the labeled DP (A). Radiolabeled proteins and nucleotides/DNA were detected by autoradiography. Priming was done in the presence of TMgNK buffer and [α- 32 P]dGTP (A, lanes 1 and 2; B, lanes 5 and 6) or TMnNK buffer and [α- 32 P]dGTP plus the unlabeled dCTP, TTP, and dATP (A, lanes 3 and 4; B, lanes 7 and 8). (C) [α- 32 P]dGTP stock was mock (lane 4) or apyrase treated (lane 5). The DP priming product obtained in TMgNK buffer and [α- 32 P]dGTP was either mock treated (lane 2) or Tdp2 treated (lane 3), which released dGMP from the DP-dGMP phosphotyrosyl linkage. Samples were resolved on a urea–20% polyacrylamide gel. The positions of 32 P-labeled 10-nucleotide marker (Invitrogen) (B) and DNA oligomers (dTG, dTGA, and dTGAA in panels B and C) are indicated, as are the positions of dGTP and dGMP. (D) HPLC analysis of dGTP and dGMP. (Panel 1) UV ( A 260 ) detection showing retention times of unlabeled dGMP and dGTP. (Panel 2) Detection of 32 P radioactivity from mock-treated DP priming products (−Tdp2), showing the absence of dGMP and the presence of residual dGTP substrate input. (Panel 3) Detection of 32 P radioactivity from Tdp2-treated DP priming products (+Tdp2), showing the presence of dGMP released by Tdp2 from DP and again some residual dGTP substrate input. The positions of dGMP and dGTP are indicated.

Techniques Used: Purification, SDS Page, Labeling, Autoradiography, Marker, High Performance Liquid Chromatography, Radioactivity

Detection of  in vitro  protein priming by purified HP. Priming reactions were performed by incubating immunoaffinity-purified HP with TMgNK buffer and [α- 32 P]dGTP (A to  C ) or another labeled nucleotide as indicated (D and E). After priming, the beads were washed, and the labeled HP was resolved on an SDS–12.5% polyacrylamide gel. A priming reaction was also performed with the DHBV MiniRT2 (DP) in TMnNK buffer and resolved on the same gel for comparison (A, lane 1). Labeled HP and DP priming products were detected by autoradiography after SDS-PAGE. (A)  In vitro  priming reactions with WT (lanes 3 and 4) or mutant (lanes 5 and 6) HP with (lanes 4 to 6) or without Hε (lane 3) coexpression in cells. GFP + Hε (lane 2) represents priming using the control purification product from cells cotransfected with GFP and the Hε-expressing plasmid. (B) After protein priming, primed HP was untreated (−; lane 1) or treated with DNase I (D; lane 2) or pronase (P; lane 3) before analysis by SDS-PAGE. (C) The purified HP was mock treated (lane 1) or RNase treated (lane 2) before being used in protein priming. Labeled HP was detected by autoradiography after SDS-PAGE (top), and HP protein levels were measured by Western blotting using the anti-FLAG (α-Flag) antibody (bottom). (D) HP purified either with (lanes 5 to 8) or without (lanes 1 to 4) the coexpressed Hε was assayed for priming activity in the presence of [α- 32 P]dGTP (G; lanes 2 and 6), [α- 32 P]TTP (T; lanes 1 and 5), [α- 32 P]dCTP (C; lanes 3 and 7), or [α- 32 P]dATP (A; lanes 4 and 8). Priming signals were quantified via phosphorimaging, normalized to the highest signal (dGTP priming, set as 100%), and denoted below the lane numbers (as a percentage of dGTP signal). The labeled HP and DP priming products are indicated. (E) Shown on the top is a schematic diagram of the mutant Hε RNAs, with the last 4 nucleotides of the internal bulge and part of the upper stem, including its bottom A-U base pair. In Hε-B6G (left), the last (6th) bulge residue (i.e., B6) was changed (from rC in the WT) to rG and in Hε-B6A (right), the same residue was changed to rA. The mutated residues are highlighted in bold. Shown at the bottom are priming products obtained with the mutant Hε RNAs. The Hε-B6G (lanes 1 and 2) or -B6A (lanes 3 and 4) mutant was coexpressed with HP, and the purified HP-Hε complex was assayed for protein priming  in vitro  in the presence of the indicated  32 P-labeled nucleotide. The labeled HP priming products are indicated, as is the position of the protein molecular mass marker (in kDa).
Figure Legend Snippet: Detection of in vitro protein priming by purified HP. Priming reactions were performed by incubating immunoaffinity-purified HP with TMgNK buffer and [α- 32 P]dGTP (A to C ) or another labeled nucleotide as indicated (D and E). After priming, the beads were washed, and the labeled HP was resolved on an SDS–12.5% polyacrylamide gel. A priming reaction was also performed with the DHBV MiniRT2 (DP) in TMnNK buffer and resolved on the same gel for comparison (A, lane 1). Labeled HP and DP priming products were detected by autoradiography after SDS-PAGE. (A) In vitro priming reactions with WT (lanes 3 and 4) or mutant (lanes 5 and 6) HP with (lanes 4 to 6) or without Hε (lane 3) coexpression in cells. GFP + Hε (lane 2) represents priming using the control purification product from cells cotransfected with GFP and the Hε-expressing plasmid. (B) After protein priming, primed HP was untreated (−; lane 1) or treated with DNase I (D; lane 2) or pronase (P; lane 3) before analysis by SDS-PAGE. (C) The purified HP was mock treated (lane 1) or RNase treated (lane 2) before being used in protein priming. Labeled HP was detected by autoradiography after SDS-PAGE (top), and HP protein levels were measured by Western blotting using the anti-FLAG (α-Flag) antibody (bottom). (D) HP purified either with (lanes 5 to 8) or without (lanes 1 to 4) the coexpressed Hε was assayed for priming activity in the presence of [α- 32 P]dGTP (G; lanes 2 and 6), [α- 32 P]TTP (T; lanes 1 and 5), [α- 32 P]dCTP (C; lanes 3 and 7), or [α- 32 P]dATP (A; lanes 4 and 8). Priming signals were quantified via phosphorimaging, normalized to the highest signal (dGTP priming, set as 100%), and denoted below the lane numbers (as a percentage of dGTP signal). The labeled HP and DP priming products are indicated. (E) Shown on the top is a schematic diagram of the mutant Hε RNAs, with the last 4 nucleotides of the internal bulge and part of the upper stem, including its bottom A-U base pair. In Hε-B6G (left), the last (6th) bulge residue (i.e., B6) was changed (from rC in the WT) to rG and in Hε-B6A (right), the same residue was changed to rA. The mutated residues are highlighted in bold. Shown at the bottom are priming products obtained with the mutant Hε RNAs. The Hε-B6G (lanes 1 and 2) or -B6A (lanes 3 and 4) mutant was coexpressed with HP, and the purified HP-Hε complex was assayed for protein priming in vitro in the presence of the indicated 32 P-labeled nucleotide. The labeled HP priming products are indicated, as is the position of the protein molecular mass marker (in kDa).

Techniques Used: In Vitro, Purification, Labeling, Autoradiography, SDS Page, Mutagenesis, Expressing, Plasmid Preparation, Western Blot, Activity Assay, Marker

Differentiation of priming initiation from DNA polymerization by S1 nuclease digestion. (A) Protein priming was conducted with DP bound to M2 affinity beads in TMnNK buffer, in the presence of [α- 32 P]dGTP and unlabeled dCTP, dATP, and TTP. Priming products were either mock treated (−; lanes 5 and 6) or S1 treated (+; lanes 7 and 8), followed by mock treatment (−; lanes 5 and 7) or Tdp2 treatment (+; lanes 6 and 8), as described in Materials and Methods. Released nucleotides or DNAs were resolved by urea-PAGE and detected by autoradiography. The 10-nucleotide marker, the dTG, dTGA, and dTGAA DNA oligomers, and dGMP positions are indicated, as is the priming initiation product (I; i.e., the single dGMP residue released by Tdp2 from DP) or polymerization products (P; DNA polymerization from the first dGMP residue). (B) Protein priming was performed with DP in TMnNK buffer with [α- 32 P]dGTP (lanes 1 and 2) or with unlabeled dGTP (unlabled dNTP denoted by parentheses) followed by the addition of [α- 32 P]TTP to extend the unlabeled DP-dGMP initiation product (lanes 3 and 4). The priming products were then mock treated (−; lanes 1 and 3) or treated with S1 nuclease (+; lanes 2 and 4), resolved by SDS-PAGE, and detected by autoradiography. (C) Priming was performed with DP (lanes 1 and 2) or HP (lanes 3 to 6) in TMgNK buffer with [α- 32 P]dGTP (lanes 1 to 4) or with unlabeled dGTP first followed by addition of [α- 32 P]dATP to extend the unlabeled HP-dGMP initiation product (lanes 5 and 6). The priming products were either mock treated (−; lanes 1, 3, and 5) or S1 treated (+; lanes 2, 4, and 6), resolved by SDS-PAGE, and detected by autoradiography. (D) The percent decreases in DP and HP priming signals as a result of S1 nuclease treatment are represented. Mock-treated DP initiation reaction in the presence of [α- 32 P]dGTP alone, with either TMnNK or TMgNK buffer, was set as 100%, and the other reaction conditions, as explained in panels B and C, were normalized to this. The decrease in priming signal due to proteolytic degradation (unrelated to S1 nuclease cleavage of internucleotide linkages) was subtracted from the calculations. (E) DP or HP was incubated with or without S1 nuclease as described above. Protease degradation was monitored by Western blotting using the M2 anti-Flag antibody. HC, antibody heavy chain. The symbol * in panels B, C, and E represents DP and HP degradation products caused by contaminating protease activity in S1. Note that only some proteolytic degradation products detected by the Western blot (E) appeared to match the  32 P-labeled degradation products (B and C) since the labeled products must have contained the priming site(s), whereas the Western blot detected only fragments containing the N-terminal FLAG tag. Also, some labeled degradation products might be present at such low levels that they were undetectable by Western blotting. Note also that the appearance of the proteolytic degradation products was accompanied by the decrease of the full-length HP or DP in panels B, C, and E. (F) The diagram depicts the cleavage of the internucleotide linkages, but not the HP-dGMP linkage, by S1.
Figure Legend Snippet: Differentiation of priming initiation from DNA polymerization by S1 nuclease digestion. (A) Protein priming was conducted with DP bound to M2 affinity beads in TMnNK buffer, in the presence of [α- 32 P]dGTP and unlabeled dCTP, dATP, and TTP. Priming products were either mock treated (−; lanes 5 and 6) or S1 treated (+; lanes 7 and 8), followed by mock treatment (−; lanes 5 and 7) or Tdp2 treatment (+; lanes 6 and 8), as described in Materials and Methods. Released nucleotides or DNAs were resolved by urea-PAGE and detected by autoradiography. The 10-nucleotide marker, the dTG, dTGA, and dTGAA DNA oligomers, and dGMP positions are indicated, as is the priming initiation product (I; i.e., the single dGMP residue released by Tdp2 from DP) or polymerization products (P; DNA polymerization from the first dGMP residue). (B) Protein priming was performed with DP in TMnNK buffer with [α- 32 P]dGTP (lanes 1 and 2) or with unlabeled dGTP (unlabled dNTP denoted by parentheses) followed by the addition of [α- 32 P]TTP to extend the unlabeled DP-dGMP initiation product (lanes 3 and 4). The priming products were then mock treated (−; lanes 1 and 3) or treated with S1 nuclease (+; lanes 2 and 4), resolved by SDS-PAGE, and detected by autoradiography. (C) Priming was performed with DP (lanes 1 and 2) or HP (lanes 3 to 6) in TMgNK buffer with [α- 32 P]dGTP (lanes 1 to 4) or with unlabeled dGTP first followed by addition of [α- 32 P]dATP to extend the unlabeled HP-dGMP initiation product (lanes 5 and 6). The priming products were either mock treated (−; lanes 1, 3, and 5) or S1 treated (+; lanes 2, 4, and 6), resolved by SDS-PAGE, and detected by autoradiography. (D) The percent decreases in DP and HP priming signals as a result of S1 nuclease treatment are represented. Mock-treated DP initiation reaction in the presence of [α- 32 P]dGTP alone, with either TMnNK or TMgNK buffer, was set as 100%, and the other reaction conditions, as explained in panels B and C, were normalized to this. The decrease in priming signal due to proteolytic degradation (unrelated to S1 nuclease cleavage of internucleotide linkages) was subtracted from the calculations. (E) DP or HP was incubated with or without S1 nuclease as described above. Protease degradation was monitored by Western blotting using the M2 anti-Flag antibody. HC, antibody heavy chain. The symbol * in panels B, C, and E represents DP and HP degradation products caused by contaminating protease activity in S1. Note that only some proteolytic degradation products detected by the Western blot (E) appeared to match the 32 P-labeled degradation products (B and C) since the labeled products must have contained the priming site(s), whereas the Western blot detected only fragments containing the N-terminal FLAG tag. Also, some labeled degradation products might be present at such low levels that they were undetectable by Western blotting. Note also that the appearance of the proteolytic degradation products was accompanied by the decrease of the full-length HP or DP in panels B, C, and E. (F) The diagram depicts the cleavage of the internucleotide linkages, but not the HP-dGMP linkage, by S1.

Techniques Used: Polyacrylamide Gel Electrophoresis, Autoradiography, Marker, SDS Page, Incubation, Western Blot, Activity Assay, Labeling, FLAG-tag

Analysis of HP protein priming products by Tdp2 cleavage of the phosphotyrosyl bond between DNA and protein. Purified HP bound to M2 antibody affinity beads was assayed for protein priming. Free nucleotides were then removed with extensive washing, and priming products were mock treated (−) or treated with Tdp2 (+) to cleave the phosphotyrosyl-DNA linkages between HP and the linked nucleotides or DNA oligomers. The supernatant, which contained the released nucleotides/DNA, was collected and resolved on a urea–20% polyacrylamide gel (B to D). The beads, which contained the primed HP, were processed for SDS-PAGE to visualize the labeled HP (A). Radiolabeled proteins and nucleotides/DNA were detected by autoradiography. Priming was done in the presence of [α- 32 P]dGTP (A, lanes 1 and 2; B, lanes 3 and 4), [α- 32 P]dATP (A, lanes 3 and 4; B, lanes 5 and 6), [α- 32 P]dGTP plus [α- 32 P]dATP (A, lanes 5 and 6; B, lanes 1 and 2; D, lanes 1 and 2), [α- 32 P]dGTP plus [α- 32 P]dTTP (D, lanes 3 and 4), [α- 32 P]dGTP plus unlabeled dATP (C, lanes 3 and 4), or the other three unlabeled dNTPs (C, lanes 5 and 6; denoted as N). Unlabeled dNTPs are denoted with parentheses in panel C. The positions of the  32 P-labeled 10-nucleotide marker (Invitrogen) (C) and DNA oligomers (dGA, dGAA, and dGAAA in panels B to D and dTG, dTGA, and dTGAA in panel C) are indicated, as are the positions of dGTP and dGMP. (E) The top diagram depicts the HP priming product, i.e., the dGAA DNA oligomer that is covalently attached to HP via Y63 and templated by the last three nucleotides (rUUC) of the internal bulge of Hε. Part of the upper stem of Hε, with its bottom A-U base pair, is also shown. The phosphotyrosyl protein-DNA linkage is specifically cleaved by Tdp2 as shown. The bottom diagram depicts DNA strand elongation following primer transfer, whereby the HP-dGAA complex is translocated from Hε to DR1, and the dGAA oligomer is further extended, potentially up to dGAAAAA in the presence of only dGTP and dATP. The putative dGAAAA or dGAAAAA product released by Tdp2 from HP is also denoted by “GAAAA(?)” in panel D.
Figure Legend Snippet: Analysis of HP protein priming products by Tdp2 cleavage of the phosphotyrosyl bond between DNA and protein. Purified HP bound to M2 antibody affinity beads was assayed for protein priming. Free nucleotides were then removed with extensive washing, and priming products were mock treated (−) or treated with Tdp2 (+) to cleave the phosphotyrosyl-DNA linkages between HP and the linked nucleotides or DNA oligomers. The supernatant, which contained the released nucleotides/DNA, was collected and resolved on a urea–20% polyacrylamide gel (B to D). The beads, which contained the primed HP, were processed for SDS-PAGE to visualize the labeled HP (A). Radiolabeled proteins and nucleotides/DNA were detected by autoradiography. Priming was done in the presence of [α- 32 P]dGTP (A, lanes 1 and 2; B, lanes 3 and 4), [α- 32 P]dATP (A, lanes 3 and 4; B, lanes 5 and 6), [α- 32 P]dGTP plus [α- 32 P]dATP (A, lanes 5 and 6; B, lanes 1 and 2; D, lanes 1 and 2), [α- 32 P]dGTP plus [α- 32 P]dTTP (D, lanes 3 and 4), [α- 32 P]dGTP plus unlabeled dATP (C, lanes 3 and 4), or the other three unlabeled dNTPs (C, lanes 5 and 6; denoted as N). Unlabeled dNTPs are denoted with parentheses in panel C. The positions of the 32 P-labeled 10-nucleotide marker (Invitrogen) (C) and DNA oligomers (dGA, dGAA, and dGAAA in panels B to D and dTG, dTGA, and dTGAA in panel C) are indicated, as are the positions of dGTP and dGMP. (E) The top diagram depicts the HP priming product, i.e., the dGAA DNA oligomer that is covalently attached to HP via Y63 and templated by the last three nucleotides (rUUC) of the internal bulge of Hε. Part of the upper stem of Hε, with its bottom A-U base pair, is also shown. The phosphotyrosyl protein-DNA linkage is specifically cleaved by Tdp2 as shown. The bottom diagram depicts DNA strand elongation following primer transfer, whereby the HP-dGAA complex is translocated from Hε to DR1, and the dGAA oligomer is further extended, potentially up to dGAAAAA in the presence of only dGTP and dATP. The putative dGAAAA or dGAAAAA product released by Tdp2 from HP is also denoted by “GAAAA(?)” in panel D.

Techniques Used: Purification, SDS Page, Labeling, Autoradiography, Marker

Related Articles

Binding Assay:

Article Title: Detection of allelic variations of human gene expression by polymerase colonies
Article Snippet: .. For each sample, 50 μl solution containing approximately 5 units of Klenow large fragment (New England Biolabs), 3 μg of single stranded binding protein (US Biochemicals), and either 0.5 μM Cy5-labeled dATP or dGTP (PerkinElmer) was pipetted onto the gel. ..

Synthesized:

Article Title: A cancer-associated point mutation disables the steric gate of human PrimPol
Article Snippet: DNA and RNA oligonucleotides were synthesized by Sigma Aldrich (St Louis, MO, USA). .. Radiolabeled nucleotides [γ-32 P] ATP , [α-32 P]dATP and [α-32 P]dGTP (3000 Ci/mmol) were obtained from Perkin Elmer (Waltham, MA, USA).

Article Title: The Zn-finger domain of human PrimPol is required to stabilize the initiating nucleotide during DNA priming
Article Snippet: Oligonucleotides and nucleotides Conventional DNA oligonucleotides were synthesized by Sigma Aldrich (St Louis, MO, USA). pppA GT and its control pA GT were obtained from IDT (Coralville, IO, USA). .. Radiolabeled nucleotides [γ-32 P]ATP , [α-32 P]dGTP, [γ-32 P]GTP and [α-32 P]dTTP (250 μCi; 3000 Ci/mmol,) were purchased from Perkin Elmer (Waltham, MA, USA).

Article Title: Human PrimPol activity is enhanced by RPA
Article Snippet: DNA oligonucleotides were synthesized by Sigma Aldrich (St Louis, Mo, USA). .. Radiolabeled nucleotides [γ-32 P]ATP and [α-32 P]dGTP (250 µCi; 3000 Ci/mmol) were obtained from Perkin Elmer (Waltham, MA, USA).

Autoradiography:

Article Title: In Vitro Epsilon RNA-Dependent Protein Priming Activity of Human Hepatitis B Virus Polymerase
Article Snippet: One microliter of [α-32 P]dGTP (10 mCi/ml [3,000 Ci/mmol]; PerkinElmer) was then added, and the reaction mixtures were incubated at 25°C for 4 h with shaking. .. The washed beads were then boiled in 2× SDS sample buffer for 10 min. Radiolabeled HP as a result of protein priming was resolved by running the eluate on an SDS–12.5% polyacrylamide gel and detected by autoradiography.

Telomerase Activity Assay:

Article Title: Multiple DNA-binding sites in Tetrahymena telomerase
Article Snippet: .. In vitro telomerase activity assay Activity of recombinant telomerase was measured by incubating 5 μl of eluted telomerase in a 10 μl reaction including 1× Telomerase buffer (50 mM Tris–Cl, pH 8.3, 1.25 mM MgCl2 , 5 mM DTT), 100 μM dTTP and 10 μM [α-32 P]dGTP at 80 Ci/mmol (0.2 μl of non-radioactive dGTP at 487 μM and 0.8 μl of [α-32 P]dGTP at 10 mCi/ml, 3000 Ci/mmol; PerkinElmer Life Sciences). .. The reactions were incubated at 30°C for 15 min (which is within the linear phase of the reaction) and then terminated by adding 90 μl of TES (50 mM Tris–HCl pH 8.3, 20 mM EDTA and 0.2% SDS).

Article Title: RNA/DNA hybrid binding affinity determines telomerase template-translocation efficiency
Article Snippet: Paragraph title: Conventional telomerase activity assay ... In brief, 2–3 μl of in vitro reconstituted telomerase was assayed in a 10-μl reaction containing 1 × telomerase reaction buffer, 1 mM dTTP, 1 mM dATP, 2 μM dGTP, 0.165 μM α-32 P-dGTP (3000 Ci/mmol, 10 mCi/ml, Perkin-Elmer) and 1 μM (TTAGGG)3 DNA primer.

Electrophoresis:

Article Title: Detection of allelic variations of human gene expression by polymerase colonies
Article Snippet: Hybridization and in situ sequencing After electrophoresis, the polony slides were washed 4 × in wash 1E (0.1 M Tris-Cl, pH 7.5, 20 mM EDTA, 0.5 M KCl) to prepare for hybridization of the sequencing primer. .. For each sample, 50 μl solution containing approximately 5 units of Klenow large fragment (New England Biolabs), 3 μg of single stranded binding protein (US Biochemicals), and either 0.5 μM Cy5-labeled dATP or dGTP (PerkinElmer) was pipetted onto the gel.

Incubation:

Article Title: In Vitro Epsilon RNA-Dependent Protein Priming Activity of Human Hepatitis B Virus Polymerase
Article Snippet: .. One microliter of [α-32 P]dGTP (10 mCi/ml [3,000 Ci/mmol]; PerkinElmer) was then added, and the reaction mixtures were incubated at 25°C for 4 h with shaking. ..

Article Title: Multiple DNA-binding sites in Tetrahymena telomerase
Article Snippet: In vitro telomerase activity assay Activity of recombinant telomerase was measured by incubating 5 μl of eluted telomerase in a 10 μl reaction including 1× Telomerase buffer (50 mM Tris–Cl, pH 8.3, 1.25 mM MgCl2 , 5 mM DTT), 100 μM dTTP and 10 μM [α-32 P]dGTP at 80 Ci/mmol (0.2 μl of non-radioactive dGTP at 487 μM and 0.8 μl of [α-32 P]dGTP at 10 mCi/ml, 3000 Ci/mmol; PerkinElmer Life Sciences). .. The reactions were incubated at 30°C for 15 min (which is within the linear phase of the reaction) and then terminated by adding 90 μl of TES (50 mM Tris–HCl pH 8.3, 20 mM EDTA and 0.2% SDS).

Article Title: Modulation of base excision repair of 8-oxoguanine by the nucleotide sequence
Article Snippet: The excision products were 3′-end labelled with [α-32 P]-dGTP (PerkinElmer, Rodgau, Germany), whose specific activity was preliminary adjusted to 300 Ci/mmol with cold dGTP. .. DNA (1 µg) was incubated with 2 units T4 DNA polymerase (37°C, 5 min) in BSA-supplemented NEBuffer 2 containing 100 µM each dATP, dCTP and dTTP, followed by addition of 20 μCi [α-32 P]-dGTP.

Article Title: RNA/DNA hybrid binding affinity determines telomerase template-translocation efficiency
Article Snippet: In brief, 2–3 μl of in vitro reconstituted telomerase was assayed in a 10-μl reaction containing 1 × telomerase reaction buffer, 1 mM dTTP, 1 mM dATP, 2 μM dGTP, 0.165 μM α-32 P-dGTP (3000 Ci/mmol, 10 mCi/ml, Perkin-Elmer) and 1 μM (TTAGGG)3 DNA primer. .. The reaction was incubated at 30°C for 60 min and terminated by phenol/chloroform extraction followed by ethanol precipitation.

Article Title: RNA/DNA hybrid binding affinity determines telomerase template-translocation efficiency
Article Snippet: .. The single-translocation reaction was initiated by adding 20 μl of incubated enzyme–primer mixture and 20 μl of pre-chilled nucleotide mixture containing 2 mM dATP, 4 μM dGTP, 0.66 μM α-32 P-dGTP (3000 Ci/mmol, 10 mCi/ml, Perkin-Elmer) and 2 μM competitive DNA primer 5′-(TTAGGG)3 -3′-amine in 1 × low-Mg2+ telomerase reaction buffer. .. Reaction was carried out on ice to slow down enzyme kinetics.

Article Title: Potency and Stereoselectivity of Cyclopropavir Triphosphate Action on Human Cytomegalovirus DNA Polymerase
Article Snippet: Briefly, all reactions were performed in 10-μl volumes and reaction mixtures contained 0.25 μM unlabeled hairpin primer template T1 (Integrated DNA Technologies), 6 nM HCMV Pol, 0.5 μM dGTP containing 0.1 μCi [α-32 P]dGTP (PerkinElmer), (+)-CPV-TP with concentrations ranging from 0 to 200 μM or (−)-CPV-TP with concentrations ranging from 0 to 500 μM, 50 mM Tris (pH 8.0), 1 mM dithiothreitol (DTT), 100 mM KCl, and 40 μg/ml bovine serum albumin (BSA). .. Reactions were initiated by adding MgCl2 to 10 mM and quenched using 10 μl of stopping buffer (0.05% bromophenol blue, 0.05% xylene cyanol, and 10 mM EDTA in formamide) after incubation at 37°C for 10 min.

Article Title: In Vitro Epsilon RNA-Dependent Protein Priming Activity of Human Hepatitis B Virus Polymerase
Article Snippet: One microliter of [α-32 P]dGTP (10 mCi/ml [3,000 Ci/mmol]; PerkinElmer) was then added, and the reaction mixtures were incubated at 25°C for 4 h with shaking. .. To test the nucleotide specificity of in vitro HBV priming, priming assays were performed using 1 μl [α-32 P]dCTP, [α-32 P]dATP, [α-32 P]TTP, or [α-32 P]dGTP (10 mCi/ml [3,000 Ci/mmol]; PerkinElmer).

Activity Assay:

Article Title: Multiple DNA-binding sites in Tetrahymena telomerase
Article Snippet: .. In vitro telomerase activity assay Activity of recombinant telomerase was measured by incubating 5 μl of eluted telomerase in a 10 μl reaction including 1× Telomerase buffer (50 mM Tris–Cl, pH 8.3, 1.25 mM MgCl2 , 5 mM DTT), 100 μM dTTP and 10 μM [α-32 P]dGTP at 80 Ci/mmol (0.2 μl of non-radioactive dGTP at 487 μM and 0.8 μl of [α-32 P]dGTP at 10 mCi/ml, 3000 Ci/mmol; PerkinElmer Life Sciences). .. The reactions were incubated at 30°C for 15 min (which is within the linear phase of the reaction) and then terminated by adding 90 μl of TES (50 mM Tris–HCl pH 8.3, 20 mM EDTA and 0.2% SDS).

Article Title: Modulation of base excision repair of 8-oxoguanine by the nucleotide sequence
Article Snippet: .. The excision products were 3′-end labelled with [α-32 P]-dGTP (PerkinElmer, Rodgau, Germany), whose specific activity was preliminary adjusted to 300 Ci/mmol with cold dGTP. .. DNA (1 µg) was incubated with 2 units T4 DNA polymerase (37°C, 5 min) in BSA-supplemented NEBuffer 2 containing 100 µM each dATP, dCTP and dTTP, followed by addition of 20 μCi [α-32 P]-dGTP.

Article Title: Telomeric G-quadruplexes are a substrate and site of localization for human telomerase
Article Snippet: .. Telomerase activity assays The following reaction was prepared to give 15 μl per sample: between 1 nM and 2 μM of the specified oligonucleotide, 50 mM Tris-HCl pH 8.5, 1 mM MgCl2 , 5 mM DTT, 1 mM spermidine-HCl, 0.5 mM dTTP, 0.5 mM dATP, 4.6 μM non-radioactive dGTP and 0.33 μM [α-32 P]dGTP at 20 mCi ml−1 , 6,000 Ci mmol−1 (PerkinElmer Life Sciences), 10% glycerol and either 150 mM KCl for experiments with 7GGT or 2.5 mM SrCl2 for experiments with 22GGG. .. The reaction was initiated by adding 5 μl of purified human telomerase, and incubating at 30 °C for 1 h. The reaction was quenched by the addition of 80 μl of stop-buffer (50 mM Tris-HCl, pH 8.3, 20 mM EDTA and 0.2% SDS) and 1–2 × 103 c.p.m. of a 5′-32 P-labelled synthetic 100-mer DNA as an internal recovery standard.

Western Blot:

Article Title: A cancer-associated point mutation disables the steric gate of human PrimPol
Article Snippet: Radiolabeled nucleotides [γ-32 P] ATP , [α-32 P]dATP and [α-32 P]dGTP (3000 Ci/mmol) were obtained from Perkin Elmer (Waltham, MA, USA). .. Secondary antibody ECLTM Anti-Rabbit IgG was detected by LuminataTM Forte Western HRP Substrate in MEFS (GE Healthcare).

Hybridization:

Article Title: Detection of allelic variations of human gene expression by polymerase colonies
Article Snippet: Paragraph title: Hybridization and in situ sequencing ... For each sample, 50 μl solution containing approximately 5 units of Klenow large fragment (New England Biolabs), 3 μg of single stranded binding protein (US Biochemicals), and either 0.5 μM Cy5-labeled dATP or dGTP (PerkinElmer) was pipetted onto the gel.

Protease Inhibitor:

Article Title: In Vitro Epsilon RNA-Dependent Protein Priming Activity of Human Hepatitis B Virus Polymerase
Article Snippet: TMgNK buffer (20 mM Tris-HCl [pH 7.0], 15 mM NaCl, 10 mM KCl, 4 mM MgCl2 ) with 1× EDTA-free protease inhibitor cocktail (Roche), 4 mM DTT, 1 mM PMSF, and 1 U RNasin Plus RNase inhibitor per μl buffer was added to the beads (19 μl per aliquot). .. One microliter of [α-32 P]dGTP (10 mCi/ml [3,000 Ci/mmol]; PerkinElmer) was then added, and the reaction mixtures were incubated at 25°C for 4 h with shaking.

Article Title: Slow mitochondrial repair of 5′-AMP renders mtDNA susceptible to damage in APTX deficient cells
Article Snippet: [γ-32 P] ATP, [α-32 P]dCTP, and [α-32 P]dGTP were from Perkin Elmer. .. Complete Protease inhibitor was from Roche.

In Vitro:

Article Title: In Vitro Epsilon RNA-Dependent Protein Priming Activity of Human Hepatitis B Virus Polymerase
Article Snippet: Paragraph title: In vitro HP priming assay. ... One microliter of [α-32 P]dGTP (10 mCi/ml [3,000 Ci/mmol]; PerkinElmer) was then added, and the reaction mixtures were incubated at 25°C for 4 h with shaking.

Article Title: Multiple DNA-binding sites in Tetrahymena telomerase
Article Snippet: .. In vitro telomerase activity assay Activity of recombinant telomerase was measured by incubating 5 μl of eluted telomerase in a 10 μl reaction including 1× Telomerase buffer (50 mM Tris–Cl, pH 8.3, 1.25 mM MgCl2 , 5 mM DTT), 100 μM dTTP and 10 μM [α-32 P]dGTP at 80 Ci/mmol (0.2 μl of non-radioactive dGTP at 487 μM and 0.8 μl of [α-32 P]dGTP at 10 mCi/ml, 3000 Ci/mmol; PerkinElmer Life Sciences). .. The reactions were incubated at 30°C for 15 min (which is within the linear phase of the reaction) and then terminated by adding 90 μl of TES (50 mM Tris–HCl pH 8.3, 20 mM EDTA and 0.2% SDS).

Article Title: RNA/DNA hybrid binding affinity determines telomerase template-translocation efficiency
Article Snippet: .. In brief, 2–3 μl of in vitro reconstituted telomerase was assayed in a 10-μl reaction containing 1 × telomerase reaction buffer, 1 mM dTTP, 1 mM dATP, 2 μM dGTP, 0.165 μM α-32 P-dGTP (3000 Ci/mmol, 10 mCi/ml, Perkin-Elmer) and 1 μM (TTAGGG)3 DNA primer. .. The reaction was incubated at 30°C for 60 min and terminated by phenol/chloroform extraction followed by ethanol precipitation.

Article Title: RNA/DNA hybrid binding affinity determines telomerase template-translocation efficiency
Article Snippet: Immuno-purified telomerase reconstituted in vitro from N-FLAG-hTERT, hTR32-195 (pseudoknot PK-1) and hTR 239-328 (CR4/5) was incubated with 0.2 μM telomeric DNA primer 5′-TT(AGGGTT)3 -3′ in 1 × low-Mg2+ telomerase reaction buffer (50 mM Tris–HCl pH 8.3, 50 mM KCl, 2 mM DTT, 1 mM MgCl2 and 1 mM spermidine) at room temperature for 10 min and then on ice for 1 min. .. The single-translocation reaction was initiated by adding 20 μl of incubated enzyme–primer mixture and 20 μl of pre-chilled nucleotide mixture containing 2 mM dATP, 4 μM dGTP, 0.66 μM α-32 P-dGTP (3000 Ci/mmol, 10 mCi/ml, Perkin-Elmer) and 2 μM competitive DNA primer 5′-(TTAGGG)3 -3′-amine in 1 × low-Mg2+ telomerase reaction buffer.

Article Title: In Vitro Epsilon RNA-Dependent Protein Priming Activity of Human Hepatitis B Virus Polymerase
Article Snippet: .. To test the nucleotide specificity of in vitro HBV priming, priming assays were performed using 1 μl [α-32 P]dCTP, [α-32 P]dATP, [α-32 P]TTP, or [α-32 P]dGTP (10 mCi/ml [3,000 Ci/mmol]; PerkinElmer). ..

Oligonucleotide Labeling:

Article Title: The Zn-finger domain of human PrimPol is required to stabilize the initiating nucleotide during DNA priming
Article Snippet: Radiolabeled nucleotides [γ-32 P]ATP , [α-32 P]dGTP, [γ-32 P]GTP and [α-32 P]dTTP (250 μCi; 3000 Ci/mmol,) were purchased from Perkin Elmer (Waltham, MA, USA). .. T4 polynucleotide kinase, used for 5′ oligonucleotide labeling, was supplied by New England Biolabs (Ipswich, MA, USA).

Generated:

Article Title: A cancer-associated point mutation disables the steric gate of human PrimPol
Article Snippet: Radiolabeled nucleotides [γ-32 P] ATP , [α-32 P]dATP and [α-32 P]dGTP (3000 Ci/mmol) were obtained from Perkin Elmer (Waltham, MA, USA). .. Anti-human PrimPol antibody (1:1000 dilution) was generated by ThermoFisher (Waltham, MA, USA).

Sequencing:

Article Title: Detection of allelic variations of human gene expression by polymerase colonies
Article Snippet: Paragraph title: Hybridization and in situ sequencing ... For each sample, 50 μl solution containing approximately 5 units of Klenow large fragment (New England Biolabs), 3 μg of single stranded binding protein (US Biochemicals), and either 0.5 μM Cy5-labeled dATP or dGTP (PerkinElmer) was pipetted onto the gel.

Recombinant:

Article Title: Multiple DNA-binding sites in Tetrahymena telomerase
Article Snippet: .. In vitro telomerase activity assay Activity of recombinant telomerase was measured by incubating 5 μl of eluted telomerase in a 10 μl reaction including 1× Telomerase buffer (50 mM Tris–Cl, pH 8.3, 1.25 mM MgCl2 , 5 mM DTT), 100 μM dTTP and 10 μM [α-32 P]dGTP at 80 Ci/mmol (0.2 μl of non-radioactive dGTP at 487 μM and 0.8 μl of [α-32 P]dGTP at 10 mCi/ml, 3000 Ci/mmol; PerkinElmer Life Sciences). .. The reactions were incubated at 30°C for 15 min (which is within the linear phase of the reaction) and then terminated by adding 90 μl of TES (50 mM Tris–HCl pH 8.3, 20 mM EDTA and 0.2% SDS).

Fluorescence:

Article Title: Detection of allelic variations of human gene expression by polymerase colonies
Article Snippet: For each sample, 50 μl solution containing approximately 5 units of Klenow large fragment (New England Biolabs), 3 μg of single stranded binding protein (US Biochemicals), and either 0.5 μM Cy5-labeled dATP or dGTP (PerkinElmer) was pipetted onto the gel. .. The gels were then washed in Wash1E to reduce background fluorescence and scanned on the ScanArray5000 with the appropriate lasers and filters.

Labeling:

Article Title: A cancer-associated point mutation disables the steric gate of human PrimPol
Article Snippet: Radiolabeled nucleotides [γ-32 P] ATP , [α-32 P]dATP and [α-32 P]dGTP (3000 Ci/mmol) were obtained from Perkin Elmer (Waltham, MA, USA). .. T4 polynucleotide kinase used for 5′ labeling of oligonucleotides was supplied by New England Biolabs (Ipswich, MA, USA).

Article Title: Human PrimPol activity is enhanced by RPA
Article Snippet: Radiolabeled nucleotides [γ-32 P]ATP and [α-32 P]dGTP (250 µCi; 3000 Ci/mmol) were obtained from Perkin Elmer (Waltham, MA, USA). .. T4 polynucleotide kinase used for 5′oligonucleotide labeling, was supplied by New England Biolabs (Ipswich, MA, USA).

Article Title: Detection of allelic variations of human gene expression by polymerase colonies
Article Snippet: The SNP was sequenced using a single base extension of the sequencing primer with fluorescently labeled dNTPs. .. For each sample, 50 μl solution containing approximately 5 units of Klenow large fragment (New England Biolabs), 3 μg of single stranded binding protein (US Biochemicals), and either 0.5 μM Cy5-labeled dATP or dGTP (PerkinElmer) was pipetted onto the gel.

Purification:

Article Title: In Vitro Epsilon RNA-Dependent Protein Priming Activity of Human Hepatitis B Virus Polymerase
Article Snippet: FLAG lysis buffer from HP purification was removed from aliquots of the HP-bound beads. .. One microliter of [α-32 P]dGTP (10 mCi/ml [3,000 Ci/mmol]; PerkinElmer) was then added, and the reaction mixtures were incubated at 25°C for 4 h with shaking.

Article Title: Telomeric G-quadruplexes are a substrate and site of localization for human telomerase
Article Snippet: Telomerase activity assays The following reaction was prepared to give 15 μl per sample: between 1 nM and 2 μM of the specified oligonucleotide, 50 mM Tris-HCl pH 8.5, 1 mM MgCl2 , 5 mM DTT, 1 mM spermidine-HCl, 0.5 mM dTTP, 0.5 mM dATP, 4.6 μM non-radioactive dGTP and 0.33 μM [α-32 P]dGTP at 20 mCi ml−1 , 6,000 Ci mmol−1 (PerkinElmer Life Sciences), 10% glycerol and either 150 mM KCl for experiments with 7GGT or 2.5 mM SrCl2 for experiments with 22GGG. .. The reaction was initiated by adding 5 μl of purified human telomerase, and incubating at 30 °C for 1 h. The reaction was quenched by the addition of 80 μl of stop-buffer (50 mM Tris-HCl, pH 8.3, 20 mM EDTA and 0.2% SDS) and 1–2 × 103 c.p.m. of a 5′-32 P-labelled synthetic 100-mer DNA as an internal recovery standard.

Article Title: Bacillus subtilis RarA modulates replication restart
Article Snippet: The radioactive nucleotides, [γ-32 P]-ATP, [α-32 P]-dCTP and [α-32 P]-dGTP, were from Perkin Elmer. .. The B. subtilis SsbA, SsbB and the SsbBA chimera (previously termed SsbB*) were purified as described ( ).

Plasmid Preparation:

Article Title: Modulation of base excision repair of 8-oxoguanine by the nucleotide sequence
Article Snippet: Plasmid DNA was isopropanol precipitated, washed with 70% ethanol and dissolved in Tris–HCl (pH 8.0). .. The excision products were 3′-end labelled with [α-32 P]-dGTP (PerkinElmer, Rodgau, Germany), whose specific activity was preliminary adjusted to 300 Ci/mmol with cold dGTP.

Software:

Article Title: Potency and Stereoselectivity of Cyclopropavir Triphosphate Action on Human Cytomegalovirus DNA Polymerase
Article Snippet: Briefly, all reactions were performed in 10-μl volumes and reaction mixtures contained 0.25 μM unlabeled hairpin primer template T1 (Integrated DNA Technologies), 6 nM HCMV Pol, 0.5 μM dGTP containing 0.1 μCi [α-32 P]dGTP (PerkinElmer), (+)-CPV-TP with concentrations ranging from 0 to 200 μM or (−)-CPV-TP with concentrations ranging from 0 to 500 μM, 50 mM Tris (pH 8.0), 1 mM dithiothreitol (DTT), 100 mM KCl, and 40 μg/ml bovine serum albumin (BSA). .. IC50 s were determined using GraphPad Prism version 6 software.

Positron Emission Tomography:

Article Title: Bacillus subtilis RarA modulates replication restart
Article Snippet: The radioactive nucleotides, [γ-32 P]-ATP, [α-32 P]-dCTP and [α-32 P]-dGTP, were from Perkin Elmer. .. All proteins were expressed in E. coli cells, from either pT712-, pET-, or pA1-based vectors ( , ).

In Situ:

Article Title: Detection of allelic variations of human gene expression by polymerase colonies
Article Snippet: Paragraph title: Hybridization and in situ sequencing ... For each sample, 50 μl solution containing approximately 5 units of Klenow large fragment (New England Biolabs), 3 μg of single stranded binding protein (US Biochemicals), and either 0.5 μM Cy5-labeled dATP or dGTP (PerkinElmer) was pipetted onto the gel.

Ethanol Precipitation:

Article Title: RNA/DNA hybrid binding affinity determines telomerase template-translocation efficiency
Article Snippet: In brief, 2–3 μl of in vitro reconstituted telomerase was assayed in a 10-μl reaction containing 1 × telomerase reaction buffer, 1 mM dTTP, 1 mM dATP, 2 μM dGTP, 0.165 μM α-32 P-dGTP (3000 Ci/mmol, 10 mCi/ml, Perkin-Elmer) and 1 μM (TTAGGG)3 DNA primer. .. The reaction was incubated at 30°C for 60 min and terminated by phenol/chloroform extraction followed by ethanol precipitation.

Article Title: RNA/DNA hybrid binding affinity determines telomerase template-translocation efficiency
Article Snippet: The single-translocation reaction was initiated by adding 20 μl of incubated enzyme–primer mixture and 20 μl of pre-chilled nucleotide mixture containing 2 mM dATP, 4 μM dGTP, 0.66 μM α-32 P-dGTP (3000 Ci/mmol, 10 mCi/ml, Perkin-Elmer) and 2 μM competitive DNA primer 5′-(TTAGGG)3 -3′-amine in 1 × low-Mg2+ telomerase reaction buffer. .. Reactions were terminated at specific time points (2, 60, 90 and 120 min) by phenol/chloroform extraction, followed by ethanol precipitation.

Concentration Assay:

Article Title: Potency and Stereoselectivity of Cyclopropavir Triphosphate Action on Human Cytomegalovirus DNA Polymerase
Article Snippet: Four different analyses were performed: 50% inhibitory concentration (IC50 ) determinations, measurements of apparent Ki values, measurements of apparent Km and k cat values, and assays of chain termination. .. Briefly, all reactions were performed in 10-μl volumes and reaction mixtures contained 0.25 μM unlabeled hairpin primer template T1 (Integrated DNA Technologies), 6 nM HCMV Pol, 0.5 μM dGTP containing 0.1 μCi [α-32 P]dGTP (PerkinElmer), (+)-CPV-TP with concentrations ranging from 0 to 200 μM or (−)-CPV-TP with concentrations ranging from 0 to 500 μM, 50 mM Tris (pH 8.0), 1 mM dithiothreitol (DTT), 100 mM KCl, and 40 μg/ml bovine serum albumin (BSA).

Lysis:

Article Title: In Vitro Epsilon RNA-Dependent Protein Priming Activity of Human Hepatitis B Virus Polymerase
Article Snippet: FLAG lysis buffer from HP purification was removed from aliquots of the HP-bound beads. .. One microliter of [α-32 P]dGTP (10 mCi/ml [3,000 Ci/mmol]; PerkinElmer) was then added, and the reaction mixtures were incubated at 25°C for 4 h with shaking.

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  • 99
    PerkinElmer α 32 p gtp
    YgdH binds (p)ppGpp antagonistically with magnesium. (A) Competition assay of purified YgdH protein (20 μM) binding a 1:1 mixture of ppGpp and pppGpp (2 nM [each]) in the absence or presence of EDTA. Representative DRaCALA spots and quantifications (average values for bound fractions and standards errors of the means [SEM]) of binding signals are shown. (B) Thin-layer chromatography (TLC) of DRaCALA binding reactions determined by using 1.5 M K 2 HPO 4 (pH 3.4) as the mobile phase. Binding reactions performed with purified MutT, Der, or YgdH were run in parallel with standards of [α- 32 <t>P]GTP</t> and a mixture of [α- 32 P]ppGpp and [α- 32 P]pppGpp (2 nM [each]). (C) Binding curves and K d determinations for YgdH interacting with α- 32 P-labeled ppGpp, pppGpp, and GTP (2 nM [each]) without or with 1.5 mM MgCl 2 . The apparent K d values corresponding to each protein-ligand interaction are shown. (D) Magnesium (0 to 10.15 mM) IC 50 determinations of binding of [α- 32 P]ppGpp (2 nM) to YgdH (50 μM). IC 50 values are shown. (E) Competition assay of YgdH (50 μM) binding [α- 32 P]ppGpp (2 nM) in the presence of 100 μM cold competitors [including (p)ppGpp and the substrates of YgdH (GMP, AMP, and IMP)] without or with 1.5 mM magnesium.
    α 32 P Gtp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 99/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    PerkinElmer γ 32 p atp
    PXR is phosphorylated in vitro and in cells (A) His-PXR (1 or 2.5 µg) was incubated at 37°C for 30 min with Cdk2 and cyclin E along with [γ- 32 <t>P]-ATP.</t> Samples were resolved on a 4–12% gradient gel, and [γ- 32 P]-ATP incorporation was visualized using a phosphor screen (upper panel), and protein amounts in the samples were detected by SimplyBlue staining of the gel (lower panel). Histone H1 and His-tag were used as a positive and negative substrate control, respectively. The PXR band was indicated with an arrow. (B) Phosphorylation sites identified by using mass spectrometry analysis in His-PXR WT phosphorylated by Cdk2/cyclin E in vitro , and in Flag-PXR WT, Flag-PXR T133A, or Flag-PXR T135A immunoprecipitated from HEK293T cells transiently transfected with corresponding plasmid ( in vivo ). Serine or threonine residues followed by an asterisk (*) indicate phosphorylated residues; UM = unmodified peptide; M = phosphorylated peptide; nd = not detected; nt = not tested. Signal intensities are calculated from area under the curve for the detected precursor ions. (C) Anti-Flag immunoprecipitated samples prepared from HEK293T cells transiently overexpressing either Flag-PXR WT (lanes 1 2) or mutants Flag-PXR T133A (lanes 4 5) or Flag-PXR T135A (lanes 7 8) were resolved on gradient gel and stained using Sypro Ruby stain. (D) Modified peptide sequence TFDTTFS*HFK (asterisk indicating serine phosphorylation), was identified based on assignment of multiple product ions ( b and y ions) in the MS/MS scan of the precursor ion at M/z 665.78. The phosphorylation of serine 167 was confirmed based on the assignment of characteristic “ y-H 3 PO 4 ” ions and other ions (based on a mass loss of 97.9769 Da). (E) Extracted-ion chromatography (XIC) of wild type and mutant PXR sequences showing elution times and signal intensities for the non-modified peptide as well as the singly phosphorylated peptide. Panel (a) and (b) are derived from the immunoprecipitated T133A sample and show the TGAQPLGVQGLTEEQR and T*GAQPLGVQGLTEEQR, respectively. Panel (c) and (d) are derived from the immunoprecipitated T135A sample and show the AGTQPLGVQGLTEEQR and AGT*QPLGVQGLTEEQR, respectively. Panel (e) and (f) are derived from the immunoprecipitated PXR WT sample and show the TGTQPLGVQGLTEEQR and T*GTQPLGVQGLTEEQR/ TGT*QPLGVQGLTEEQR, respectively. Relative abundance (RA) of the signals of the corresponding peptides is noted for each XIC.
    γ 32 P Atp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 99/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 10 article reviews
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    YgdH binds (p)ppGpp antagonistically with magnesium. (A) Competition assay of purified YgdH protein (20 μM) binding a 1:1 mixture of ppGpp and pppGpp (2 nM [each]) in the absence or presence of EDTA. Representative DRaCALA spots and quantifications (average values for bound fractions and standards errors of the means [SEM]) of binding signals are shown. (B) Thin-layer chromatography (TLC) of DRaCALA binding reactions determined by using 1.5 M K 2 HPO 4 (pH 3.4) as the mobile phase. Binding reactions performed with purified MutT, Der, or YgdH were run in parallel with standards of [α- 32 P]GTP and a mixture of [α- 32 P]ppGpp and [α- 32 P]pppGpp (2 nM [each]). (C) Binding curves and K d determinations for YgdH interacting with α- 32 P-labeled ppGpp, pppGpp, and GTP (2 nM [each]) without or with 1.5 mM MgCl 2 . The apparent K d values corresponding to each protein-ligand interaction are shown. (D) Magnesium (0 to 10.15 mM) IC 50 determinations of binding of [α- 32 P]ppGpp (2 nM) to YgdH (50 μM). IC 50 values are shown. (E) Competition assay of YgdH (50 μM) binding [α- 32 P]ppGpp (2 nM) in the presence of 100 μM cold competitors [including (p)ppGpp and the substrates of YgdH (GMP, AMP, and IMP)] without or with 1.5 mM magnesium.

    Journal: mBio

    Article Title: Novel (p)ppGpp Binding and Metabolizing Proteins of Escherichia coli

    doi: 10.1128/mBio.02188-17

    Figure Lengend Snippet: YgdH binds (p)ppGpp antagonistically with magnesium. (A) Competition assay of purified YgdH protein (20 μM) binding a 1:1 mixture of ppGpp and pppGpp (2 nM [each]) in the absence or presence of EDTA. Representative DRaCALA spots and quantifications (average values for bound fractions and standards errors of the means [SEM]) of binding signals are shown. (B) Thin-layer chromatography (TLC) of DRaCALA binding reactions determined by using 1.5 M K 2 HPO 4 (pH 3.4) as the mobile phase. Binding reactions performed with purified MutT, Der, or YgdH were run in parallel with standards of [α- 32 P]GTP and a mixture of [α- 32 P]ppGpp and [α- 32 P]pppGpp (2 nM [each]). (C) Binding curves and K d determinations for YgdH interacting with α- 32 P-labeled ppGpp, pppGpp, and GTP (2 nM [each]) without or with 1.5 mM MgCl 2 . The apparent K d values corresponding to each protein-ligand interaction are shown. (D) Magnesium (0 to 10.15 mM) IC 50 determinations of binding of [α- 32 P]ppGpp (2 nM) to YgdH (50 μM). IC 50 values are shown. (E) Competition assay of YgdH (50 μM) binding [α- 32 P]ppGpp (2 nM) in the presence of 100 μM cold competitors [including (p)ppGpp and the substrates of YgdH (GMP, AMP, and IMP)] without or with 1.5 mM magnesium.

    Article Snippet: 32 P-labeled pppGpp was synthesized from [α-32 P]GTP (PerkinElmer) by incubating 125 nM [α-32 P]GTP with 4 μM purified RelSeq (1–285)-His protein ( ) in buffer S (containing 25 mM Tris [pH 9.0], 100 mM NaCl, 15 mM MgCl2 , and 8 mM ATP) at 37°C for 1 h. The sample was subsequently incubated for 5 min at 95°C to stop synthesis, and the denatured RelSeq (1–285)-His protein was removed by centrifugation at 13,400 rpm for 10 min at 4°C.

    Techniques: Competitive Binding Assay, Purification, Binding Assay, Thin Layer Chromatography, Labeling

    Translational GTPases are conserved targets of (p)ppGpp. (A) Competition assay of RF3 and Der (20 μM) and LepA (10 μM) binding [α- 32 P]ppGpp (2 nM) in the presence of cold competitors (100 μM). (B and C) Binding curves and K d determination of RF3 (B) and Der (C) binding of α- 32 P-labeled ppGpp, pppGpp, GTP, or GDP (2 nM [each]). At least three replicates were performed. The apparent K d values corresponding to each protein-ligand interaction are shown. (D) Dissociation curves for Der (50 μM) and [α- 32 P]ppGpp (2 nM) in the presence of either ppGpp or GTP (100 μM) (cold). (E and F) Binding curves and K d determination for DerG1 (E) and SaDer (F) binding α- 32 P-labeled ppGpp, pppGpp, or GTP (2 nM [each]). At least three replicates were performed. The apparent K d values are shown for each protein-ligand interaction.

    Journal: mBio

    Article Title: Novel (p)ppGpp Binding and Metabolizing Proteins of Escherichia coli

    doi: 10.1128/mBio.02188-17

    Figure Lengend Snippet: Translational GTPases are conserved targets of (p)ppGpp. (A) Competition assay of RF3 and Der (20 μM) and LepA (10 μM) binding [α- 32 P]ppGpp (2 nM) in the presence of cold competitors (100 μM). (B and C) Binding curves and K d determination of RF3 (B) and Der (C) binding of α- 32 P-labeled ppGpp, pppGpp, GTP, or GDP (2 nM [each]). At least three replicates were performed. The apparent K d values corresponding to each protein-ligand interaction are shown. (D) Dissociation curves for Der (50 μM) and [α- 32 P]ppGpp (2 nM) in the presence of either ppGpp or GTP (100 μM) (cold). (E and F) Binding curves and K d determination for DerG1 (E) and SaDer (F) binding α- 32 P-labeled ppGpp, pppGpp, or GTP (2 nM [each]). At least three replicates were performed. The apparent K d values are shown for each protein-ligand interaction.

    Article Snippet: 32 P-labeled pppGpp was synthesized from [α-32 P]GTP (PerkinElmer) by incubating 125 nM [α-32 P]GTP with 4 μM purified RelSeq (1–285)-His protein ( ) in buffer S (containing 25 mM Tris [pH 9.0], 100 mM NaCl, 15 mM MgCl2 , and 8 mM ATP) at 37°C for 1 h. The sample was subsequently incubated for 5 min at 95°C to stop synthesis, and the denatured RelSeq (1–285)-His protein was removed by centrifugation at 13,400 rpm for 10 min at 4°C.

    Techniques: Competitive Binding Assay, Binding Assay, Labeling

    HypB specifically binds (p)ppGpp with physiological affinity. (A) Competition assay of HypB (20 μM) binding α- 32 P-labeled ppGpp and pppGpp (2 nM [each]) in the presence of cold competitors (100 μM). (B) Binding curves and K d determination for HypB binding α- 32 P-labeled ppGpp, pppGpp, and GTP (2 nM [each]). Three replicates were performed, and the apparent K d values are indicated.

    Journal: mBio

    Article Title: Novel (p)ppGpp Binding and Metabolizing Proteins of Escherichia coli

    doi: 10.1128/mBio.02188-17

    Figure Lengend Snippet: HypB specifically binds (p)ppGpp with physiological affinity. (A) Competition assay of HypB (20 μM) binding α- 32 P-labeled ppGpp and pppGpp (2 nM [each]) in the presence of cold competitors (100 μM). (B) Binding curves and K d determination for HypB binding α- 32 P-labeled ppGpp, pppGpp, and GTP (2 nM [each]). Three replicates were performed, and the apparent K d values are indicated.

    Article Snippet: 32 P-labeled pppGpp was synthesized from [α-32 P]GTP (PerkinElmer) by incubating 125 nM [α-32 P]GTP with 4 μM purified RelSeq (1–285)-His protein ( ) in buffer S (containing 25 mM Tris [pH 9.0], 100 mM NaCl, 15 mM MgCl2 , and 8 mM ATP) at 37°C for 1 h. The sample was subsequently incubated for 5 min at 95°C to stop synthesis, and the denatured RelSeq (1–285)-His protein was removed by centrifugation at 13,400 rpm for 10 min at 4°C.

    Techniques: Competitive Binding Assay, Binding Assay, Labeling

    In vitro cleavage of ppGpp by MutT, NudG, NadR, and TrmE. (A) Competition assay of whole-cell lysates containing overexpressed MutT and NudG binding [α- 32 P]ppGpp (2 nM) in the presence of cold competitors (100 μM). (B) Competition assay of purified NadR (left) and TrmE (right) (20 μM [each]) binding α- 32 P-labeled ppGpp and pppGpp (2 nM [each]) in the presence of cold competitors (100 μM). (C) DRaCALA spots of purified proteins (10 μM) binding a mixture of α- 32 P-labeled ppGpp and pppGpp (2 nM [each]) in the absence or presence of EDTA (25 mM). (D) TLC assessment of cleavage products from the binding reactions described for panel C. A mixture of ppGpp and pppGpp was run as the standard, and both molecules are indicated. (E) Quantification of (p)ppGpp percentage determined as described for panel D. (F) Competition assay of whole-cell lysates containing overproduced MutT and NudG binding [α- 32 P](p)ppGpp (2 nM) in the presence of cold competitors and their native substrates (100 μM [each]). Representative DRaCALA spots are shown. 8OdG, 8-oxo-dGTP; 8OG, 8-oxo-GTP; 2OdA, 2-hydroxyl-dATP; 2OA, 2-hydroxyl-ATP. (G) TLC assessment of cleavage products of [α- 32 P]ppGpp (10 nM) determined using purified MutT and NudG (1 μM) in the presence of cold competitors (100 μM) or excess EDTA (25 mM). Samples were incubated at 30°C for 10 min (or 1 h; see Fig. S6D ), and reactions were stopped by addition of excess EDTA (25 mM). pGp and ppGpp are indicated.

    Journal: mBio

    Article Title: Novel (p)ppGpp Binding and Metabolizing Proteins of Escherichia coli

    doi: 10.1128/mBio.02188-17

    Figure Lengend Snippet: In vitro cleavage of ppGpp by MutT, NudG, NadR, and TrmE. (A) Competition assay of whole-cell lysates containing overexpressed MutT and NudG binding [α- 32 P]ppGpp (2 nM) in the presence of cold competitors (100 μM). (B) Competition assay of purified NadR (left) and TrmE (right) (20 μM [each]) binding α- 32 P-labeled ppGpp and pppGpp (2 nM [each]) in the presence of cold competitors (100 μM). (C) DRaCALA spots of purified proteins (10 μM) binding a mixture of α- 32 P-labeled ppGpp and pppGpp (2 nM [each]) in the absence or presence of EDTA (25 mM). (D) TLC assessment of cleavage products from the binding reactions described for panel C. A mixture of ppGpp and pppGpp was run as the standard, and both molecules are indicated. (E) Quantification of (p)ppGpp percentage determined as described for panel D. (F) Competition assay of whole-cell lysates containing overproduced MutT and NudG binding [α- 32 P](p)ppGpp (2 nM) in the presence of cold competitors and their native substrates (100 μM [each]). Representative DRaCALA spots are shown. 8OdG, 8-oxo-dGTP; 8OG, 8-oxo-GTP; 2OdA, 2-hydroxyl-dATP; 2OA, 2-hydroxyl-ATP. (G) TLC assessment of cleavage products of [α- 32 P]ppGpp (10 nM) determined using purified MutT and NudG (1 μM) in the presence of cold competitors (100 μM) or excess EDTA (25 mM). Samples were incubated at 30°C for 10 min (or 1 h; see Fig. S6D ), and reactions were stopped by addition of excess EDTA (25 mM). pGp and ppGpp are indicated.

    Article Snippet: 32 P-labeled pppGpp was synthesized from [α-32 P]GTP (PerkinElmer) by incubating 125 nM [α-32 P]GTP with 4 μM purified RelSeq (1–285)-His protein ( ) in buffer S (containing 25 mM Tris [pH 9.0], 100 mM NaCl, 15 mM MgCl2 , and 8 mM ATP) at 37°C for 1 h. The sample was subsequently incubated for 5 min at 95°C to stop synthesis, and the denatured RelSeq (1–285)-His protein was removed by centrifugation at 13,400 rpm for 10 min at 4°C.

    Techniques: In Vitro, Competitive Binding Assay, Binding Assay, Purification, Labeling, Thin Layer Chromatography, Incubation

    GTP biosynthesis and salvage pathways are targeted by (p)ppGpp. (A) Schematic of purine biosynthesis pathways with (p)ppGpp targets highlighted by colored boxes. Green indicates E. coli targets identified here; blue indicates specific Bacillus / Staphylococcus targets; red indicates E. coli targets reported previously but not confirmed in this study; gray indicates a target found in E. coli , Bacillus , and Staphylococcus . G, guanine; X, xanthine; H, hypoxanthine; A, adenine; PRPP, phosphoribosyl pyrophosphate; Gln, glutamine. (B) Binding curves and apparent K d values for E. coli Gpt, Hpt, and Apt binding pppGpp and ppGpp (2 nM [each]). The average values for bound fractions and standard errors of the means (SEM) determined for at least three replicates were plotted and the curve-fitted and K d values determined as previously described ( 37 ). The apparent K d values corresponding to each protein-ligand interaction are shown. (C) Competition assay of Gpt, Hpt, and Apt (20 μM [each]) binding [α- 32 P]ppGpp (2 nM) in the presence of cold competitors (100 μM). The average values for bound fractions and standard errors of the means (SEM) determined for at least three replicates were plotted. Representative DRaCALA spots are shown above the respective diagrams.

    Journal: mBio

    Article Title: Novel (p)ppGpp Binding and Metabolizing Proteins of Escherichia coli

    doi: 10.1128/mBio.02188-17

    Figure Lengend Snippet: GTP biosynthesis and salvage pathways are targeted by (p)ppGpp. (A) Schematic of purine biosynthesis pathways with (p)ppGpp targets highlighted by colored boxes. Green indicates E. coli targets identified here; blue indicates specific Bacillus / Staphylococcus targets; red indicates E. coli targets reported previously but not confirmed in this study; gray indicates a target found in E. coli , Bacillus , and Staphylococcus . G, guanine; X, xanthine; H, hypoxanthine; A, adenine; PRPP, phosphoribosyl pyrophosphate; Gln, glutamine. (B) Binding curves and apparent K d values for E. coli Gpt, Hpt, and Apt binding pppGpp and ppGpp (2 nM [each]). The average values for bound fractions and standard errors of the means (SEM) determined for at least three replicates were plotted and the curve-fitted and K d values determined as previously described ( 37 ). The apparent K d values corresponding to each protein-ligand interaction are shown. (C) Competition assay of Gpt, Hpt, and Apt (20 μM [each]) binding [α- 32 P]ppGpp (2 nM) in the presence of cold competitors (100 μM). The average values for bound fractions and standard errors of the means (SEM) determined for at least three replicates were plotted. Representative DRaCALA spots are shown above the respective diagrams.

    Article Snippet: 32 P-labeled pppGpp was synthesized from [α-32 P]GTP (PerkinElmer) by incubating 125 nM [α-32 P]GTP with 4 μM purified RelSeq (1–285)-His protein ( ) in buffer S (containing 25 mM Tris [pH 9.0], 100 mM NaCl, 15 mM MgCl2 , and 8 mM ATP) at 37°C for 1 h. The sample was subsequently incubated for 5 min at 95°C to stop synthesis, and the denatured RelSeq (1–285)-His protein was removed by centrifugation at 13,400 rpm for 10 min at 4°C.

    Techniques: Binding Assay, Competitive Binding Assay

    The balance between the poly(A) length and the downstream post-poly(A) length affects deadenylation efficiency. ( A ) Schematic representation of a series of Mini- let-7 reporter constructs. These reporter RNAs include four let-7 target sites and were capped with [α- 32 P] GTP. The subscripts show the length of an RNA sequence including the shown base. The asterisks indicate a radiolabel. ( B ) Deadenylation assay for Mini- let-7 reporter RNAs with S2 cell lysate overexpressed Ago1. ( C ) The signal intensity of the bands in B was quantified, and the ratio of the intensity from the lower band for the total intensity was calculated and plotted. Mini- let-7 -A 20 effectively shifted to A 0 . While the length of the post-poly(A) sequence negatively correlated with the deadenylation efficiency, elongating the internal poly(A) sequence improved. The graph shows means and standard deviations ( n = 3). ( D ) Deadenylation assay for Mini- let-7 -A 60 C 10 and Mini- let-7 -N 40 A 20 C 10 with S2 cell lysate overexpressed Ago1. ( E ) The signal intensity of the bands in D was quantified, and the ratio of the intensity from the lower band for the total intensity was calculated and plotted. Mini- let-7 -A 60 C 10 was deadenylated more effectively than Mini- let-7 -N 40 A 20 C 10 . The graph shows means and standard deviations ( n = 3).

    Journal: RNA

    Article Title: CCR4 and CAF1 deadenylases have an intrinsic activity to remove the post-poly(A) sequence

    doi: 10.1261/rna.057679.116

    Figure Lengend Snippet: The balance between the poly(A) length and the downstream post-poly(A) length affects deadenylation efficiency. ( A ) Schematic representation of a series of Mini- let-7 reporter constructs. These reporter RNAs include four let-7 target sites and were capped with [α- 32 P] GTP. The subscripts show the length of an RNA sequence including the shown base. The asterisks indicate a radiolabel. ( B ) Deadenylation assay for Mini- let-7 reporter RNAs with S2 cell lysate overexpressed Ago1. ( C ) The signal intensity of the bands in B was quantified, and the ratio of the intensity from the lower band for the total intensity was calculated and plotted. Mini- let-7 -A 20 effectively shifted to A 0 . While the length of the post-poly(A) sequence negatively correlated with the deadenylation efficiency, elongating the internal poly(A) sequence improved. The graph shows means and standard deviations ( n = 3). ( D ) Deadenylation assay for Mini- let-7 -A 60 C 10 and Mini- let-7 -N 40 A 20 C 10 with S2 cell lysate overexpressed Ago1. ( E ) The signal intensity of the bands in D was quantified, and the ratio of the intensity from the lower band for the total intensity was calculated and plotted. Mini- let-7 -A 60 C 10 was deadenylated more effectively than Mini- let-7 -N 40 A 20 C 10 . The graph shows means and standard deviations ( n = 3).

    Article Snippet: RNAs were radiolabeled by using ScriptCap m7 G Capping System (CELLSCRIPT) and [α-32 P] GTP (PerkinElmer).

    Techniques: Construct, Sequencing

    PXR is phosphorylated in vitro and in cells (A) His-PXR (1 or 2.5 µg) was incubated at 37°C for 30 min with Cdk2 and cyclin E along with [γ- 32 P]-ATP. Samples were resolved on a 4–12% gradient gel, and [γ- 32 P]-ATP incorporation was visualized using a phosphor screen (upper panel), and protein amounts in the samples were detected by SimplyBlue staining of the gel (lower panel). Histone H1 and His-tag were used as a positive and negative substrate control, respectively. The PXR band was indicated with an arrow. (B) Phosphorylation sites identified by using mass spectrometry analysis in His-PXR WT phosphorylated by Cdk2/cyclin E in vitro , and in Flag-PXR WT, Flag-PXR T133A, or Flag-PXR T135A immunoprecipitated from HEK293T cells transiently transfected with corresponding plasmid ( in vivo ). Serine or threonine residues followed by an asterisk (*) indicate phosphorylated residues; UM = unmodified peptide; M = phosphorylated peptide; nd = not detected; nt = not tested. Signal intensities are calculated from area under the curve for the detected precursor ions. (C) Anti-Flag immunoprecipitated samples prepared from HEK293T cells transiently overexpressing either Flag-PXR WT (lanes 1 2) or mutants Flag-PXR T133A (lanes 4 5) or Flag-PXR T135A (lanes 7 8) were resolved on gradient gel and stained using Sypro Ruby stain. (D) Modified peptide sequence TFDTTFS*HFK (asterisk indicating serine phosphorylation), was identified based on assignment of multiple product ions ( b and y ions) in the MS/MS scan of the precursor ion at M/z 665.78. The phosphorylation of serine 167 was confirmed based on the assignment of characteristic “ y-H 3 PO 4 ” ions and other ions (based on a mass loss of 97.9769 Da). (E) Extracted-ion chromatography (XIC) of wild type and mutant PXR sequences showing elution times and signal intensities for the non-modified peptide as well as the singly phosphorylated peptide. Panel (a) and (b) are derived from the immunoprecipitated T133A sample and show the TGAQPLGVQGLTEEQR and T*GAQPLGVQGLTEEQR, respectively. Panel (c) and (d) are derived from the immunoprecipitated T135A sample and show the AGTQPLGVQGLTEEQR and AGT*QPLGVQGLTEEQR, respectively. Panel (e) and (f) are derived from the immunoprecipitated PXR WT sample and show the TGTQPLGVQGLTEEQR and T*GTQPLGVQGLTEEQR/ TGT*QPLGVQGLTEEQR, respectively. Relative abundance (RA) of the signals of the corresponding peptides is noted for each XIC.

    Journal: Biochemical pharmacology

    Article Title: Identification and Characterization of Phosphorylation Sites within the Pregnane X Receptor Protein

    doi: 10.1016/j.bcp.2013.10.015

    Figure Lengend Snippet: PXR is phosphorylated in vitro and in cells (A) His-PXR (1 or 2.5 µg) was incubated at 37°C for 30 min with Cdk2 and cyclin E along with [γ- 32 P]-ATP. Samples were resolved on a 4–12% gradient gel, and [γ- 32 P]-ATP incorporation was visualized using a phosphor screen (upper panel), and protein amounts in the samples were detected by SimplyBlue staining of the gel (lower panel). Histone H1 and His-tag were used as a positive and negative substrate control, respectively. The PXR band was indicated with an arrow. (B) Phosphorylation sites identified by using mass spectrometry analysis in His-PXR WT phosphorylated by Cdk2/cyclin E in vitro , and in Flag-PXR WT, Flag-PXR T133A, or Flag-PXR T135A immunoprecipitated from HEK293T cells transiently transfected with corresponding plasmid ( in vivo ). Serine or threonine residues followed by an asterisk (*) indicate phosphorylated residues; UM = unmodified peptide; M = phosphorylated peptide; nd = not detected; nt = not tested. Signal intensities are calculated from area under the curve for the detected precursor ions. (C) Anti-Flag immunoprecipitated samples prepared from HEK293T cells transiently overexpressing either Flag-PXR WT (lanes 1 2) or mutants Flag-PXR T133A (lanes 4 5) or Flag-PXR T135A (lanes 7 8) were resolved on gradient gel and stained using Sypro Ruby stain. (D) Modified peptide sequence TFDTTFS*HFK (asterisk indicating serine phosphorylation), was identified based on assignment of multiple product ions ( b and y ions) in the MS/MS scan of the precursor ion at M/z 665.78. The phosphorylation of serine 167 was confirmed based on the assignment of characteristic “ y-H 3 PO 4 ” ions and other ions (based on a mass loss of 97.9769 Da). (E) Extracted-ion chromatography (XIC) of wild type and mutant PXR sequences showing elution times and signal intensities for the non-modified peptide as well as the singly phosphorylated peptide. Panel (a) and (b) are derived from the immunoprecipitated T133A sample and show the TGAQPLGVQGLTEEQR and T*GAQPLGVQGLTEEQR, respectively. Panel (c) and (d) are derived from the immunoprecipitated T135A sample and show the AGTQPLGVQGLTEEQR and AGT*QPLGVQGLTEEQR, respectively. Panel (e) and (f) are derived from the immunoprecipitated PXR WT sample and show the TGTQPLGVQGLTEEQR and T*GTQPLGVQGLTEEQR/ TGT*QPLGVQGLTEEQR, respectively. Relative abundance (RA) of the signals of the corresponding peptides is noted for each XIC.

    Article Snippet: Different amount of His-PXR as indicated (1 µg or 2.5 µg) was incubated in kinase buffer with 20 ng Cdk2/cyclin E (EMD Millipore, Billerica, MA), 5 µCi [γ-32 P]ATP (Perkin-Elmer, Santa Clara, CA), and 5 µM cold ATP.

    Techniques: In Vitro, Incubation, Staining, Mass Spectrometry, Immunoprecipitation, Transfection, Plasmid Preparation, In Vivo, Modification, Sequencing, Ion Chromatography, Mutagenesis, Derivative Assay

    5′-adenylation of long RNA substrates. ( A ) Schematic diagram of the experimental strategy. The > 100-mer RNA substrate is too long for 5′-AppRNA formation to induce a measurable gel shift relative to a 5′-monophosphate. Therefore, an appropriate 8–17 deoxyribozyme is used to cleave the 5′-portion of the RNA substrate, leaving a small fragment for which 5′-AppRNA formation does cause a gel shift. ( B ) The strategy in A applied to the 160-nt P4–P6 domain of the Tetrahymena group I intron RNA. Blocking oligos were uncapped. The three time points are at 0.5 min, 10 min, and 1 h (6% PAGE). The RNA substrate was internally radiolabeled by transcription incorporating α- 32 P-ATP; the 5′-monophosphate was provided by performing the transcription in the presence of excess GMP (see Materials and Methods). Although the side products have not been studied in great detail, the side product formed in the first experiment (P4–P6 with no DNA blocking oligo) is tentatively assigned as circularized P4–P6 on the basis of attempted 5′- 32 P-radiolabeling with T4 polynucleotide kinase and γ- 32 P-ATP; no reaction was observed alongside a positive control. Only the lower band (a mixture of 5′-monophosphate and 5′-AppRNA) was carried to the 8–17 deoxyribozyme cleavage experiment. std, P4–P6 standard RNA carried through all reactions with no blocking oligo, except that T4 RNA ligase was omitted. ( C ) The strategy in A ).

    Journal: RNA

    Article Title: Practical and general synthesis of 5?-adenylated RNA (5?-AppRNA)

    doi: 10.1261/rna.5247704

    Figure Lengend Snippet: 5′-adenylation of long RNA substrates. ( A ) Schematic diagram of the experimental strategy. The > 100-mer RNA substrate is too long for 5′-AppRNA formation to induce a measurable gel shift relative to a 5′-monophosphate. Therefore, an appropriate 8–17 deoxyribozyme is used to cleave the 5′-portion of the RNA substrate, leaving a small fragment for which 5′-AppRNA formation does cause a gel shift. ( B ) The strategy in A applied to the 160-nt P4–P6 domain of the Tetrahymena group I intron RNA. Blocking oligos were uncapped. The three time points are at 0.5 min, 10 min, and 1 h (6% PAGE). The RNA substrate was internally radiolabeled by transcription incorporating α- 32 P-ATP; the 5′-monophosphate was provided by performing the transcription in the presence of excess GMP (see Materials and Methods). Although the side products have not been studied in great detail, the side product formed in the first experiment (P4–P6 with no DNA blocking oligo) is tentatively assigned as circularized P4–P6 on the basis of attempted 5′- 32 P-radiolabeling with T4 polynucleotide kinase and γ- 32 P-ATP; no reaction was observed alongside a positive control. Only the lower band (a mixture of 5′-monophosphate and 5′-AppRNA) was carried to the 8–17 deoxyribozyme cleavage experiment. std, P4–P6 standard RNA carried through all reactions with no blocking oligo, except that T4 RNA ligase was omitted. ( C ) The strategy in A ).

    Article Snippet: Radiolabeled RNAs were prepared with γ-32 P-ATP (PerkinElmer) and T4 PNK (New England Biolabs) and purified by 20% denaturing PAGE followed by ethanol precipitation.

    Techniques: Electrophoretic Mobility Shift Assay, Blocking Assay, Polyacrylamide Gel Electrophoresis, Radioactivity, Positive Control