Structured Review

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.
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Images

1) 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

2) 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

3) 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

4) 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

5) 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

6) 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

7) 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

8) 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

9) 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

10) 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

11) 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

12) 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

13) 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

14) 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

15) 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

16) 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

17) 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

18) 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

19) 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

20) 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

21) 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

22) 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

23) 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

24) 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

25) 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

26) 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

27) 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

28) 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

29) Product Images from "Eukaryotic Elongation Factor 1A Interacts with Sphingosine Kinase and Directly Enhances Its Catalytic Activity *"

Article Title: Eukaryotic Elongation Factor 1A Interacts with Sphingosine Kinase and Directly Enhances Its Catalytic Activity *

Journal:

doi: 10.1074/jbc.M708782200

Phosphorylation of eEF1A1 does not alter binding to SK1 or SK2. A , GST-eEF1A1 bound to GSH-Sepharose was incubated with S6K in the presence of γ- 32 P]ATP, and the phosphorylation of GST-eEF1A1 was detected using phosphorimaging ( upper panel
Figure Legend Snippet: Phosphorylation of eEF1A1 does not alter binding to SK1 or SK2. A , GST-eEF1A1 bound to GSH-Sepharose was incubated with S6K in the presence of γ- 32 P]ATP, and the phosphorylation of GST-eEF1A1 was detected using phosphorimaging ( upper panel

Techniques Used: Binding Assay, Incubation

30) Product Images from "Characterization of WZ4003 and HTH-01-015 as selective inhibitors of the LKB1-tumour-suppressor-activated NUAK kinases"

Article Title: Characterization of WZ4003 and HTH-01-015 as selective inhibitors of the LKB1-tumour-suppressor-activated NUAK kinases

Journal: Biochemical Journal

doi: 10.1042/BJ20131152

HTH-01-015, a specific NUAK1 inhibitor ( A ) Chemical structure of the NUAK1-specific inhibitor HTH-01-015. ( B ) Wild-type (WT) GST–NUAK1 and GST–NUAK2 were assayed using 200 μM Sakamototide in the presence of 100 μM [γ- 32 P]ATP (~500 c.p.m./pmol) with the indicated concentrations of HTH-01-015. The IC 50 graph was plotted using Graphpad Prism software with non-linear regression analysis. The results are presented as the percentage of kinase activity relative to the DMSO-treated control. Results are means±S.D. for triplicate reactions with similar results obtained in at least one other experiment. ( C ) Kinase profiling of the HTH-01-015 inhibitor at 1 μM was carried out against the panel of 140 kinases at the The International Centre for Protein Kinase Profiling ( http://www.kinase-screen.mrc.ac.uk/ ). AMPK family kinases are indicated with an asterisk, LKB1 with a filled hexagon and NUAK1 with an arrow. The full names of the kinases can be found in the legend to Supplementary Table S1 (at http://www.biochemj.org/bj/457/bj4570215add.htm ). ( D ) As in ( B ) except that HTH-01-015 comparative IC 50 values were derived for wild-type (WT) GST–NUAK1 and GST–NUAK1[A195T].
Figure Legend Snippet: HTH-01-015, a specific NUAK1 inhibitor ( A ) Chemical structure of the NUAK1-specific inhibitor HTH-01-015. ( B ) Wild-type (WT) GST–NUAK1 and GST–NUAK2 were assayed using 200 μM Sakamototide in the presence of 100 μM [γ- 32 P]ATP (~500 c.p.m./pmol) with the indicated concentrations of HTH-01-015. The IC 50 graph was plotted using Graphpad Prism software with non-linear regression analysis. The results are presented as the percentage of kinase activity relative to the DMSO-treated control. Results are means±S.D. for triplicate reactions with similar results obtained in at least one other experiment. ( C ) Kinase profiling of the HTH-01-015 inhibitor at 1 μM was carried out against the panel of 140 kinases at the The International Centre for Protein Kinase Profiling ( http://www.kinase-screen.mrc.ac.uk/ ). AMPK family kinases are indicated with an asterisk, LKB1 with a filled hexagon and NUAK1 with an arrow. The full names of the kinases can be found in the legend to Supplementary Table S1 (at http://www.biochemj.org/bj/457/bj4570215add.htm ). ( D ) As in ( B ) except that HTH-01-015 comparative IC 50 values were derived for wild-type (WT) GST–NUAK1 and GST–NUAK1[A195T].

Techniques Used: Software, Activity Assay, Derivative Assay

XMD-17-51, a potent semi-specific NUAK1 inhibitor ( A ) Chemical structure of XMD-17-51. ( B ) Wild-type (WT) GST–NUAK1 and GST–NUAK1[A195T] were assayed using 200 μM Sakamototide in the presence of 100 μM [γ- 32 P]ATP (~500 c.p.m./pmol) with the indicated concentrations of XMD-17-51. The IC 50 graph was plotted using Graphpad Prism software with non-linear regression analysis. The results are presented as the percentage of kinase activity relative to the DMSO-treated control. Results are means±S.D. for triplicate reactions with similar results obtained in at least one other experiment. ( C ) Kinase profiling of the XMD-17-51 inhibitor at 1 μM was carried out against the panel of 140 kinases at the The International Centre for Protein Kinase Profiling ( http://www.kinase-screen.mrc.ac.uk/ ). AMPK family kinases are indicated with an asterisk, LKB1 with a filled hexagon and NUAK1 with an arrow. The full names of the kinases can be found in the legend to Supplementary Table S1 (at http://www.biochemj.org/bj/457/bj4570215add.htm ). ( D ) HEK-293 cells were treated in the absence (DMSO) or presence of the indicated concentrations of XMD-17-51 over 16 h. Cell medium was then replaced with either normal DMEM containing no EDTA-PBS-based cell dissociation buffer (−) or EDTA-PBS-based cell dissociation buffer (+) containing the same concentration of XMD-17-51 that the cells were previously incubated in. Cell detachment was induced with gentle tapping of the plates followed by gentle centrifugation at 70 g for 3 min. Cells were lysed immediately after removal of the supernatant. Endogenous MYPT1 was immunoprecipitated from 0.5 mg of the cell lysates. The immunoprecipitates were immunoblotted for the detection of p-Ser 445 MYPT1 and total MYPT1. The cell lysates were subjected to immunoblotting for the detection of p-Ser 79 ACC and total ACC. Similar results were obtained in three separate experiments.
Figure Legend Snippet: XMD-17-51, a potent semi-specific NUAK1 inhibitor ( A ) Chemical structure of XMD-17-51. ( B ) Wild-type (WT) GST–NUAK1 and GST–NUAK1[A195T] were assayed using 200 μM Sakamototide in the presence of 100 μM [γ- 32 P]ATP (~500 c.p.m./pmol) with the indicated concentrations of XMD-17-51. The IC 50 graph was plotted using Graphpad Prism software with non-linear regression analysis. The results are presented as the percentage of kinase activity relative to the DMSO-treated control. Results are means±S.D. for triplicate reactions with similar results obtained in at least one other experiment. ( C ) Kinase profiling of the XMD-17-51 inhibitor at 1 μM was carried out against the panel of 140 kinases at the The International Centre for Protein Kinase Profiling ( http://www.kinase-screen.mrc.ac.uk/ ). AMPK family kinases are indicated with an asterisk, LKB1 with a filled hexagon and NUAK1 with an arrow. The full names of the kinases can be found in the legend to Supplementary Table S1 (at http://www.biochemj.org/bj/457/bj4570215add.htm ). ( D ) HEK-293 cells were treated in the absence (DMSO) or presence of the indicated concentrations of XMD-17-51 over 16 h. Cell medium was then replaced with either normal DMEM containing no EDTA-PBS-based cell dissociation buffer (−) or EDTA-PBS-based cell dissociation buffer (+) containing the same concentration of XMD-17-51 that the cells were previously incubated in. Cell detachment was induced with gentle tapping of the plates followed by gentle centrifugation at 70 g for 3 min. Cells were lysed immediately after removal of the supernatant. Endogenous MYPT1 was immunoprecipitated from 0.5 mg of the cell lysates. The immunoprecipitates were immunoblotted for the detection of p-Ser 445 MYPT1 and total MYPT1. The cell lysates were subjected to immunoblotting for the detection of p-Ser 79 ACC and total ACC. Similar results were obtained in three separate experiments.

Techniques Used: Software, Activity Assay, Concentration Assay, Incubation, Centrifugation, Immunoprecipitation

XMD-18-42, a semi-specific NUAK1 inhibitor ( A ) Chemical structure of XMD-18-42. ( B ) Wild-type (WT) GST–NUAK1 and GST–NUAK1[A195T] were assayed using 200 μM Sakamototide in the presence of 100 μM [γ- 32 P]ATP (~500 c.p.m./pmol) with the indicated concentrations of XMD-18-42. The IC 50 graph was plotted using Graphpad Prism software with non-linear regression analysis. The results are presented as the percentage of kinase activity relative to the DMSO-treated control. Results are means±S.D. for triplicate reactions with similar results obtained in at least one other experiment. ( C ) Kinase profiling of the XMD-18-42 inhibitor at 1 μM was carried out against the panel of 140 kinases at the The International Centre for Protein Kinase Profiling ( http://www.kinase-screen.mrc.ac.uk/ ). AMPK family kinases are indicated with an asterisk, LKB1 with a filled hexagon and NUAK1 with an arrow. The full names of the kinases can be found in the legend to Supplementary Table S1 (at http://www.biochemj.org/bj/457/bj4570215add.htm ). ( D ) HEK-293 cells were treated in the absence (DMSO) or presence of the indicated concentrations of XMD-18-42 over 16 h. Cell medium was then replaced with either normal DMEM containing no EDTA-PBS-based cell dissociation buffer (−) or EDTA-PBS-based cell dissociation buffer (+) containing the same concentration of XMD-18-42 that the cells were previously incubated in. Cell detachment was induced with gentle tapping of the plates followed by gentle centrifugation at 70 g for 3 min. Cells were lysed immediately after removal of the supernatant. Endogenous MYPT1 was immunoprecipitated from 0.5 mg of the cell lysates. The immunoprecipitates were immunoblotted for the detection of p-Ser 445 MYPT1 and total MYPT1. The cell lysates were subjected to immunoblotting for the detection of p-Ser 79 ACC and total ACC. Similar results were obtained in three separate experiments.
Figure Legend Snippet: XMD-18-42, a semi-specific NUAK1 inhibitor ( A ) Chemical structure of XMD-18-42. ( B ) Wild-type (WT) GST–NUAK1 and GST–NUAK1[A195T] were assayed using 200 μM Sakamototide in the presence of 100 μM [γ- 32 P]ATP (~500 c.p.m./pmol) with the indicated concentrations of XMD-18-42. The IC 50 graph was plotted using Graphpad Prism software with non-linear regression analysis. The results are presented as the percentage of kinase activity relative to the DMSO-treated control. Results are means±S.D. for triplicate reactions with similar results obtained in at least one other experiment. ( C ) Kinase profiling of the XMD-18-42 inhibitor at 1 μM was carried out against the panel of 140 kinases at the The International Centre for Protein Kinase Profiling ( http://www.kinase-screen.mrc.ac.uk/ ). AMPK family kinases are indicated with an asterisk, LKB1 with a filled hexagon and NUAK1 with an arrow. The full names of the kinases can be found in the legend to Supplementary Table S1 (at http://www.biochemj.org/bj/457/bj4570215add.htm ). ( D ) HEK-293 cells were treated in the absence (DMSO) or presence of the indicated concentrations of XMD-18-42 over 16 h. Cell medium was then replaced with either normal DMEM containing no EDTA-PBS-based cell dissociation buffer (−) or EDTA-PBS-based cell dissociation buffer (+) containing the same concentration of XMD-18-42 that the cells were previously incubated in. Cell detachment was induced with gentle tapping of the plates followed by gentle centrifugation at 70 g for 3 min. Cells were lysed immediately after removal of the supernatant. Endogenous MYPT1 was immunoprecipitated from 0.5 mg of the cell lysates. The immunoprecipitates were immunoblotted for the detection of p-Ser 445 MYPT1 and total MYPT1. The cell lysates were subjected to immunoblotting for the detection of p-Ser 79 ACC and total ACC. Similar results were obtained in three separate experiments.

Techniques Used: Software, Activity Assay, Concentration Assay, Incubation, Centrifugation, Immunoprecipitation

WZ4003, a specific NUAK1 and NUAK2 inhibitor ( A ) Chemical structure of the NUAK1/NUAK2 inhibitor WZ4003. ( B ) Wild-type (WT) GST–NUAK1 and GST–NUAK2 were assayed using 200 μM Sakamototide in the presence of 100 μM [γ- 32 P]ATP (~500 c.p.m./pmol) with the indicated concentrations of WZ4003. The IC 50 graph was plotted using GraphPad Prism software with non-linear regression analysis. The results are presented as the percentage of kinase activity relative to the DMSO-treated control. Results are means±S.D. for triplicate reactions with similar results obtained in at least one other experiment. ( C ) Kinase profiling of the WZ4003 inhibitor at 1 μM was carried out against the panel of 140 kinases at the The International Centre for Protein Kinase Profiling ( http://www.kinase-screen.mrc.ac.uk/ ). AMPK family kinases are indicated with an asterisk, LKB1 with a filled hexagon and NUAK1 with an arrow. The full names of the kinases can be found in the legend to Supplementary Table S1 (at http://www.biochemj.org/bj/457/bj4570215add.htm ). ( D ) Wild-type (WT) GST–NUAK1 and GST–NUAK1[A195T] were purified from HEK-293 cells following transient transfection and relative levels of wild-type and mutant enzymes were analysed by Coomassie Blue staining of a polyacrylamide gel (bottom panel). Intrinsic kinase activities of the equivalent amounts of NUAK1 and NUAK1[A195T] were compared by carrying out a quantitative kinase activity assay by calculating the relative kinase-mediated incorporation of [γ- 32 P]ATP into the Sakamototide substrate peptide. Values are means±S.D. for an experiment carried out in triplicate. ( E ) As in ( B ) except that WZ4003 comparative IC 50 values were derived for wild-type (WT) GST–NUAK1 and GST—NUAK1[A195T].
Figure Legend Snippet: WZ4003, a specific NUAK1 and NUAK2 inhibitor ( A ) Chemical structure of the NUAK1/NUAK2 inhibitor WZ4003. ( B ) Wild-type (WT) GST–NUAK1 and GST–NUAK2 were assayed using 200 μM Sakamototide in the presence of 100 μM [γ- 32 P]ATP (~500 c.p.m./pmol) with the indicated concentrations of WZ4003. The IC 50 graph was plotted using GraphPad Prism software with non-linear regression analysis. The results are presented as the percentage of kinase activity relative to the DMSO-treated control. Results are means±S.D. for triplicate reactions with similar results obtained in at least one other experiment. ( C ) Kinase profiling of the WZ4003 inhibitor at 1 μM was carried out against the panel of 140 kinases at the The International Centre for Protein Kinase Profiling ( http://www.kinase-screen.mrc.ac.uk/ ). AMPK family kinases are indicated with an asterisk, LKB1 with a filled hexagon and NUAK1 with an arrow. The full names of the kinases can be found in the legend to Supplementary Table S1 (at http://www.biochemj.org/bj/457/bj4570215add.htm ). ( D ) Wild-type (WT) GST–NUAK1 and GST–NUAK1[A195T] were purified from HEK-293 cells following transient transfection and relative levels of wild-type and mutant enzymes were analysed by Coomassie Blue staining of a polyacrylamide gel (bottom panel). Intrinsic kinase activities of the equivalent amounts of NUAK1 and NUAK1[A195T] were compared by carrying out a quantitative kinase activity assay by calculating the relative kinase-mediated incorporation of [γ- 32 P]ATP into the Sakamototide substrate peptide. Values are means±S.D. for an experiment carried out in triplicate. ( E ) As in ( B ) except that WZ4003 comparative IC 50 values were derived for wild-type (WT) GST–NUAK1 and GST—NUAK1[A195T].

Techniques Used: Software, Activity Assay, Purification, Transfection, Mutagenesis, Staining, Kinase Assay, Derivative Assay

31) Product Images from "The Extracytoplasmic Linker Peptide of the Sensor Protein SaeS Tunes the Kinase Activity Required for Staphylococcal Virulence in Response to Host Signals"

Article Title: The Extracytoplasmic Linker Peptide of the Sensor Protein SaeS Tunes the Kinase Activity Required for Staphylococcal Virulence in Response to Host Signals

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1004799

Membrane vesicles harboring the linker peptide mutant SaeS exhibit altered phosphotransferase activity. (A) Levels of SaeR-P following incubation of SaeR, [γ- 32 P] ATP, and membrane vesicles (300 μg) harboring the wild-type or the various linker mutant SaeS proteins. (B) Quantitation of the phosphotransfer assay shown in (A). Each datum of the plot depicts the level of SaeR-P relative to that by the wild-type SaeS at the initial time (1 min). Data correspond to the mean values of three independent experiments, and error bars show standard deviation.
Figure Legend Snippet: Membrane vesicles harboring the linker peptide mutant SaeS exhibit altered phosphotransferase activity. (A) Levels of SaeR-P following incubation of SaeR, [γ- 32 P] ATP, and membrane vesicles (300 μg) harboring the wild-type or the various linker mutant SaeS proteins. (B) Quantitation of the phosphotransfer assay shown in (A). Each datum of the plot depicts the level of SaeR-P relative to that by the wild-type SaeS at the initial time (1 min). Data correspond to the mean values of three independent experiments, and error bars show standard deviation.

Techniques Used: Mutagenesis, Activity Assay, Incubation, Quantitation Assay, Standard Deviation

Alanine substitutions in the linker peptide alter the kinase and phosphotransferase activities of SaeS. (A) The autokinase activity of wild type (WT) and select linker peptide mutants of SaeS. The purified MBP-SaeS proteins (5 μM) were incubated with [γ- 32 P] ATP at RT for 20 min. The autoradiograph of the phosphorylated MBP-SaeS (upper panel) is shown with its quantification results in a bar graph (lower panel). (B) Assessment of the autokinase activity of wild type (WT) and select linker peptide mutants for 30 min. The wild-type or the linker peptide mutant MBP-SaeS proteins (5 μM) were mixed with [γ- 32 P] ATP and, at the indicated times, the level of phosphorylated MBP-SaeS was analyzed by phosphor imager analysis. (C) Quantitation of the autophosphorylation assays shown in (B). The plot depicts the levels of MBP-SaeS-P relative to the wild-type MBP-SaeS-P at time 1 min as a function of time. (D) Phosphotransferase activity of the wild type (WT) and select linker peptide mutants of SaeS. Phosphorylated MBP-SaeS (5 μM) was mixed with SaeR (10 μM). At the times indicated, the reaction was stopped and the phosphorylated proteins were analyzed by SDS-PAGE and phosphor imager analysis. (E) Quantification of the phosphotransfer assays shown in (D). Each datum on the plot depicts the level of SaeR-P relative to that of the wild-type SaeS at the initial time (1 min). All data correspond to the mean values of three independent experiments, and error bars show standard deviation. For statistical analyses, unpaired two-tailed student’s t-test was used. ***, p
Figure Legend Snippet: Alanine substitutions in the linker peptide alter the kinase and phosphotransferase activities of SaeS. (A) The autokinase activity of wild type (WT) and select linker peptide mutants of SaeS. The purified MBP-SaeS proteins (5 μM) were incubated with [γ- 32 P] ATP at RT for 20 min. The autoradiograph of the phosphorylated MBP-SaeS (upper panel) is shown with its quantification results in a bar graph (lower panel). (B) Assessment of the autokinase activity of wild type (WT) and select linker peptide mutants for 30 min. The wild-type or the linker peptide mutant MBP-SaeS proteins (5 μM) were mixed with [γ- 32 P] ATP and, at the indicated times, the level of phosphorylated MBP-SaeS was analyzed by phosphor imager analysis. (C) Quantitation of the autophosphorylation assays shown in (B). The plot depicts the levels of MBP-SaeS-P relative to the wild-type MBP-SaeS-P at time 1 min as a function of time. (D) Phosphotransferase activity of the wild type (WT) and select linker peptide mutants of SaeS. Phosphorylated MBP-SaeS (5 μM) was mixed with SaeR (10 μM). At the times indicated, the reaction was stopped and the phosphorylated proteins were analyzed by SDS-PAGE and phosphor imager analysis. (E) Quantification of the phosphotransfer assays shown in (D). Each datum on the plot depicts the level of SaeR-P relative to that of the wild-type SaeS at the initial time (1 min). All data correspond to the mean values of three independent experiments, and error bars show standard deviation. For statistical analyses, unpaired two-tailed student’s t-test was used. ***, p

Techniques Used: Activity Assay, Purification, Incubation, Autoradiography, Mutagenesis, Quantitation Assay, SDS Page, Standard Deviation, Two Tailed Test

32) Product Images from "Diversity of two-component systems: insights into the signal transduction mechanism by the Staphylococcus aureus two-component system GraSR"

Article Title: Diversity of two-component systems: insights into the signal transduction mechanism by the Staphylococcus aureus two-component system GraSR

Journal: F1000Research

doi: 10.12688/f1000research.5512.2

Attempt to phosphorylate GST-GraS in the presence of GraX. GST-GraS 5 µM alone or along with 2 or 5 µM GraX was incubated at room temperature for 30 min before adding [γ- 32 P] ATP. GraX (5 µM) was used as control. Reactions were quenched at different time intervals and analyzed by 12.5% SDS-PAGE. On left side are shown the radiogram images of the SDS-PAGE and on the right the Coomassie stained images of the SDS-PAGE.
Figure Legend Snippet: Attempt to phosphorylate GST-GraS in the presence of GraX. GST-GraS 5 µM alone or along with 2 or 5 µM GraX was incubated at room temperature for 30 min before adding [γ- 32 P] ATP. GraX (5 µM) was used as control. Reactions were quenched at different time intervals and analyzed by 12.5% SDS-PAGE. On left side are shown the radiogram images of the SDS-PAGE and on the right the Coomassie stained images of the SDS-PAGE.

Techniques Used: Incubation, SDS Page, Staining

Attempted autophosphorylation of GraS. ( A ) GST-GraS (5 µM) in 50 mM Tris, 50 mM KCl, 10 mM CaCl 2 , and 5 mM MgCl 2 (pH 7.4) was incubated with 10 µM γ- 32 P-ATP. The reaction was quenched at different time intervals. ( B ) Autophosphorylation of His-GraS under the same conditions as in ( A ). Left panel represent phosphor imaging and right panels represent the Coomassie staining of the SDS-PAGE gels.
Figure Legend Snippet: Attempted autophosphorylation of GraS. ( A ) GST-GraS (5 µM) in 50 mM Tris, 50 mM KCl, 10 mM CaCl 2 , and 5 mM MgCl 2 (pH 7.4) was incubated with 10 µM γ- 32 P-ATP. The reaction was quenched at different time intervals. ( B ) Autophosphorylation of His-GraS under the same conditions as in ( A ). Left panel represent phosphor imaging and right panels represent the Coomassie staining of the SDS-PAGE gels.

Techniques Used: Incubation, Imaging, Staining, SDS Page

Autophosphorylation of BceS. ( A ) GST-BceS (5 µM) was incubated with [γ- 32 P] ATP (1 mM) in 50 mM Tris, 50 mM KCl, 20 mM CaCl 2 , and 5 mM MgCl 2 (pH 7.4). Reactions were quenched at different incubation times and analyzed by 12.5% SDS-PAGE. ( B ) The experimental data obtained in ( A ) were quantified using ImageJ and plotted against the incubation time (the error bars represent the standard deviations calculated from three independent experiments). The data were fitted to the equation given in the Experimental Section.
Figure Legend Snippet: Autophosphorylation of BceS. ( A ) GST-BceS (5 µM) was incubated with [γ- 32 P] ATP (1 mM) in 50 mM Tris, 50 mM KCl, 20 mM CaCl 2 , and 5 mM MgCl 2 (pH 7.4). Reactions were quenched at different incubation times and analyzed by 12.5% SDS-PAGE. ( B ) The experimental data obtained in ( A ) were quantified using ImageJ and plotted against the incubation time (the error bars represent the standard deviations calculated from three independent experiments). The data were fitted to the equation given in the Experimental Section.

Techniques Used: Incubation, SDS Page

33) Product Images from "Intermolecular Binding between TIFA-FHA and TIFA-pT Mediates Tumor Necrosis Factor Alpha Stimulation and NF-?B Activation"

Article Title: Intermolecular Binding between TIFA-FHA and TIFA-pT Mediates Tumor Necrosis Factor Alpha Stimulation and NF-?B Activation

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00438-12

Enhancement of TIFA phosphorylation by TNF-α stimulation. (A) Schematic overview of TIFA domain structure (top) and N-terminal sequences of TIFA orthologues from different species (bottom). T9 is highlighted in dark gray. (B) MS/MS spectrum of the peptide containing pT9 in TIFA. The precursor ion 827.2816 2+ is from ADPMTSFEDAD(pT)EE. (C) NanoPro immunoassay demonstrates the phosphorylation status of overexpressed Myc-tagged WT and T9A mutant TIFA with or without TNF-α and phosphatase treatment. The peaks at pI 4.75 and pI 4.63 are assigned to unphosphorylated TIFA and singly phosphorylated TIFA, respectively. The bar graph in the lower panel represents the ratios of peak areas of singly phosphorylated TIFA to total TIFA. The results represent means ± standard deviations (SD) from at least 3 independent experiments. (D) NanoPro immunoassay of endogenous TIFA. The conditions were the same as those for panel C. The pI values differ from the peaks in panel C due to lack of the Myc tag. (E) Recombinant TIFA or the T9A mutant was incubated with [γ- 32 P]ATP and lysates from cells treated with or without TNF-α for 30 min. During incubation, alkaline phosphatase (PPtase) was added as indicated. The reaction mixtures were separated by SDS-PAGE, and the bands were revealed by autoradiography. (F) The experimental conditions were the same as those for panel E except for the inclusion of caffeine or inhibitor cocktails for AKT, IRAK, TAK, or PKC (Go 6976 and Go 6983).
Figure Legend Snippet: Enhancement of TIFA phosphorylation by TNF-α stimulation. (A) Schematic overview of TIFA domain structure (top) and N-terminal sequences of TIFA orthologues from different species (bottom). T9 is highlighted in dark gray. (B) MS/MS spectrum of the peptide containing pT9 in TIFA. The precursor ion 827.2816 2+ is from ADPMTSFEDAD(pT)EE. (C) NanoPro immunoassay demonstrates the phosphorylation status of overexpressed Myc-tagged WT and T9A mutant TIFA with or without TNF-α and phosphatase treatment. The peaks at pI 4.75 and pI 4.63 are assigned to unphosphorylated TIFA and singly phosphorylated TIFA, respectively. The bar graph in the lower panel represents the ratios of peak areas of singly phosphorylated TIFA to total TIFA. The results represent means ± standard deviations (SD) from at least 3 independent experiments. (D) NanoPro immunoassay of endogenous TIFA. The conditions were the same as those for panel C. The pI values differ from the peaks in panel C due to lack of the Myc tag. (E) Recombinant TIFA or the T9A mutant was incubated with [γ- 32 P]ATP and lysates from cells treated with or without TNF-α for 30 min. During incubation, alkaline phosphatase (PPtase) was added as indicated. The reaction mixtures were separated by SDS-PAGE, and the bands were revealed by autoradiography. (F) The experimental conditions were the same as those for panel E except for the inclusion of caffeine or inhibitor cocktails for AKT, IRAK, TAK, or PKC (Go 6976 and Go 6983).

Techniques Used: Mass Spectrometry, Mutagenesis, Recombinant, Incubation, SDS Page, Autoradiography

34) Product Images from "The PAS domains of the major sporulation kinase in Bacillus subtilis play a role in tetramer formation that is essential for the autokinase activity. The PAS domains of the major sporulation kinase in Bacillus subtilis play a role in tetramer formation that is essential for the autokinase activity"

Article Title: The PAS domains of the major sporulation kinase in Bacillus subtilis play a role in tetramer formation that is essential for the autokinase activity. The PAS domains of the major sporulation kinase in Bacillus subtilis play a role in tetramer formation that is essential for the autokinase activity

Journal: MicrobiologyOpen

doi: 10.1002/mbo3.481

In vitro autophosphorylation assay for PAS ‐A domain deletion form of KinA (KinA Δ PAS ‐A ). (a) Autophosphorylation activities of KinA and KinA Δ PAS ‐A (Δ PAS ‐A) were measured in an in vitro reaction. Each of the purified proteins was analyzed by SDS ‐ PAGE (lanes 1 and 2, 0.1 μg protein stained with Coomassie brilliant blue, CBB in panel a) and with in vitro autophosphorylation in the presence of [γ‐ 32 P] ATP followed by autoradiography of SDS ‐ PAGE (lanes 3 and 4, 32 P in panel a). For kinetic analysis, at the indicated times, aliquots were removed from the in vitro autophosphorylation reaction mixture, mixed with SDS ‐ PAGE sample buffer to stop the reaction, and analyzed by SDS ‐ PAGE followed by autoradiography (lanes 5–11). (b) Relative phosphorylation levels over time of reaction. The fractions of phosphorylation levels plotted on the y‐axis in the graph were defined as the ratio of each of the radiolabeled proteins at the indicated time point to the maximum level of that present at the end point of the reaction and are expressed as the relative level ( RL ). (c) Graph indicates the semilogarithmic plot of the value of (1‐ RL ) as a function of time. The estimated values of k obs (pseudo‐first‐order rate constant) for the autophosphorylation of KinA and KinA Δ PAS ‐A were calculated from the slopes. The symbols are the same as in (b). (d) Spo0A phosphorylation by KinA and KinA Δ PAS ‐A through phosphorelay. The purified proteins as indicated on the top were incubated with [γ‐ 32 P] ATP as described in Materials and Methods. (+) and (−) indicate with and without proteins, respectively. Whole reaction mixtures were analyzed by SDS ‐ PAGE followed by autoradiography. Each of the gel images displayed is one of the representative results (a and d). The mean from three independent experiments with standard error is shown (b and c)
Figure Legend Snippet: In vitro autophosphorylation assay for PAS ‐A domain deletion form of KinA (KinA Δ PAS ‐A ). (a) Autophosphorylation activities of KinA and KinA Δ PAS ‐A (Δ PAS ‐A) were measured in an in vitro reaction. Each of the purified proteins was analyzed by SDS ‐ PAGE (lanes 1 and 2, 0.1 μg protein stained with Coomassie brilliant blue, CBB in panel a) and with in vitro autophosphorylation in the presence of [γ‐ 32 P] ATP followed by autoradiography of SDS ‐ PAGE (lanes 3 and 4, 32 P in panel a). For kinetic analysis, at the indicated times, aliquots were removed from the in vitro autophosphorylation reaction mixture, mixed with SDS ‐ PAGE sample buffer to stop the reaction, and analyzed by SDS ‐ PAGE followed by autoradiography (lanes 5–11). (b) Relative phosphorylation levels over time of reaction. The fractions of phosphorylation levels plotted on the y‐axis in the graph were defined as the ratio of each of the radiolabeled proteins at the indicated time point to the maximum level of that present at the end point of the reaction and are expressed as the relative level ( RL ). (c) Graph indicates the semilogarithmic plot of the value of (1‐ RL ) as a function of time. The estimated values of k obs (pseudo‐first‐order rate constant) for the autophosphorylation of KinA and KinA Δ PAS ‐A were calculated from the slopes. The symbols are the same as in (b). (d) Spo0A phosphorylation by KinA and KinA Δ PAS ‐A through phosphorelay. The purified proteins as indicated on the top were incubated with [γ‐ 32 P] ATP as described in Materials and Methods. (+) and (−) indicate with and without proteins, respectively. Whole reaction mixtures were analyzed by SDS ‐ PAGE followed by autoradiography. Each of the gel images displayed is one of the representative results (a and d). The mean from three independent experiments with standard error is shown (b and c)

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

35) Product Images from "The hVps34‐ SGK3 pathway alleviates sustained PI3K/Akt inhibition by stimulating mTORC1 and tumour growth"

Article Title: The hVps34‐ SGK3 pathway alleviates sustained PI3K/Akt inhibition by stimulating mTORC1 and tumour growth

Journal: The EMBO Journal

doi: 10.15252/embj.201693929

Akt inhibition induces dose‐dependent inhibition of cell growth in BT‐474c cells in vitro and stimulates SGK3 to activate mTORC1 BT‐474c cells were treated with DMSO, 3 μM 14h and the indicated doses of MK‐2206 (A) or AZD5363 (B) inhibitors. Cell confluency was measured on the Incucyte ZOOM every 4 h for up to 6 days. BT‐474c cells were treated for 1 h or 5 days with 1 μM MK‐2206 or 3 μM 14h, as indicated. SGK3 (upper panel) and S6K1 (middle panel) were immunoprecipitated and subjected to in vitro kinase assay by measuring phosphorylation of the Crosstide substrate peptide in the presence of 0.1 mM [γ‐ 32 P]ATP in a 30 min 30°C reaction. Kinase reactions are presented as means ± SD for triplicate reaction. Immunoprecipitates (IP) were also analysed by immunoblot with the indicated antibodies. The cell lysates were subjected to immunoblot analysis using the indicated antibodies (bottom panel). Source data are available online for this figure.
Figure Legend Snippet: Akt inhibition induces dose‐dependent inhibition of cell growth in BT‐474c cells in vitro and stimulates SGK3 to activate mTORC1 BT‐474c cells were treated with DMSO, 3 μM 14h and the indicated doses of MK‐2206 (A) or AZD5363 (B) inhibitors. Cell confluency was measured on the Incucyte ZOOM every 4 h for up to 6 days. BT‐474c cells were treated for 1 h or 5 days with 1 μM MK‐2206 or 3 μM 14h, as indicated. SGK3 (upper panel) and S6K1 (middle panel) were immunoprecipitated and subjected to in vitro kinase assay by measuring phosphorylation of the Crosstide substrate peptide in the presence of 0.1 mM [γ‐ 32 P]ATP in a 30 min 30°C reaction. Kinase reactions are presented as means ± SD for triplicate reaction. Immunoprecipitates (IP) were also analysed by immunoblot with the indicated antibodies. The cell lysates were subjected to immunoblot analysis using the indicated antibodies (bottom panel). Source data are available online for this figure.

Techniques Used: Inhibition, In Vitro, Immunoprecipitation, Kinase Assay

SGK 3 counteracts inhibition of the PI 3K/Akt pathway by phosphorylating TSC 2 and stimulating mTORC 1 ZR‐75‐1 cells were treated for 1 h or 5 days with 1 μM MK‐2206, 1 μM GDC0941 or 3 μM 14h inhibitors, alone or in combination, as indicated. The cell lysates were analysed by immunoblot using the indicated antibodies. SGK3 (upper panel), Akt1 (middle panel) and S6K1 (lower panel) were immunoprecipitated from the same cell lysates and subjected to in vitro kinase assay by measuring phosphorylation of the Crosstide substrate peptide for kinases in the presence of 0.1 mM [γ‐ 32 P]ATP in a 30 min, 30°C reaction. Kinase reactions are presented as means ± SD for triplicate reaction. Immunoprecipitates (IP) were also analysed by immunoblot with the indicated antibodies. SGK3 was knocked down in ZR‐75‐1 cells by using shRNA probe B and compared to a control shRNA probe, named sh scramble. After infection, the cells were kept for 2 days in puromycin selection media and then seeded for the experiment. The cells were treated with 1 μM MK‐2206 for 1 h or 5 days. The cell lysates were subjected to immunoblot analysis with the indicated antibodies.
Figure Legend Snippet: SGK 3 counteracts inhibition of the PI 3K/Akt pathway by phosphorylating TSC 2 and stimulating mTORC 1 ZR‐75‐1 cells were treated for 1 h or 5 days with 1 μM MK‐2206, 1 μM GDC0941 or 3 μM 14h inhibitors, alone or in combination, as indicated. The cell lysates were analysed by immunoblot using the indicated antibodies. SGK3 (upper panel), Akt1 (middle panel) and S6K1 (lower panel) were immunoprecipitated from the same cell lysates and subjected to in vitro kinase assay by measuring phosphorylation of the Crosstide substrate peptide for kinases in the presence of 0.1 mM [γ‐ 32 P]ATP in a 30 min, 30°C reaction. Kinase reactions are presented as means ± SD for triplicate reaction. Immunoprecipitates (IP) were also analysed by immunoblot with the indicated antibodies. SGK3 was knocked down in ZR‐75‐1 cells by using shRNA probe B and compared to a control shRNA probe, named sh scramble. After infection, the cells were kept for 2 days in puromycin selection media and then seeded for the experiment. The cells were treated with 1 μM MK‐2206 for 1 h or 5 days. The cell lysates were subjected to immunoblot analysis with the indicated antibodies.

Techniques Used: Inhibition, Immunoprecipitation, In Vitro, Kinase Assay, shRNA, Infection, Selection

SGK 3 counteracts inhibition of the PI3K/Akt pathway by phosphorylating TSC 2 and stimulating S6K1 ZR‐75‐1 cells were treated with 1 μM MK‐2206 for the indicated times. SGK3 (upper panel) and S6K1 (middle panel) were immunoprecipitated and subjected to in vitro kinase assay by measuring phosphorylation of the Crosstide substrate peptide in the presence of 0.1 mM [γ‐ 32 P]ATP in a 30 min 30°C reaction. Kinase reactions are presented as means ± SD for triplicate reaction. Immunoprecipitates (IP) were also analysed by immunoblot with the indicated antibodies. The cell lysates were also analysed by immunoblot using the indicated antibodies (lower panel).
Figure Legend Snippet: SGK 3 counteracts inhibition of the PI3K/Akt pathway by phosphorylating TSC 2 and stimulating S6K1 ZR‐75‐1 cells were treated with 1 μM MK‐2206 for the indicated times. SGK3 (upper panel) and S6K1 (middle panel) were immunoprecipitated and subjected to in vitro kinase assay by measuring phosphorylation of the Crosstide substrate peptide in the presence of 0.1 mM [γ‐ 32 P]ATP in a 30 min 30°C reaction. Kinase reactions are presented as means ± SD for triplicate reaction. Immunoprecipitates (IP) were also analysed by immunoblot with the indicated antibodies. The cell lysates were also analysed by immunoblot using the indicated antibodies (lower panel).

Techniques Used: Inhibition, Immunoprecipitation, In Vitro, Kinase Assay

Further evidence that SGK3 activity induced by inhibition of PI3K/Akt is regulated by hVps34 ZR‐75‐1 cells were treated with 1 μM MK‐2206 for 5 days, and then, 1 h prior to cell lysis, cells were further treated with increasing doses of SAR405. ZR‐75‐1 cells cultured in serum in the absence of Akt inhibitor were treated for 1 h with the indicated concentrations of SAR405. The cell lysates were analysed by immunoblot using the indicated antibodies. ZR‐75‐1 cells were treated for 5 days with 1 μM MK‐2206 or 1 μM GDC0941. One hour prior to lysis, the cells were incubated in the presence of absence of 0.3 μM SAR405. SGK3 was immunoprecipitated from lysates and subjected to in vitro kinase assay by measuring phosphorylation of the Crosstide substrate peptide in the presence of 0.1 mM [γ‐ 32 P]ATP in a 30 min 30°C reaction (upper panel). Kinase reactions are presented as means ± SD for triplicate reaction. Immunoprecipitates (IP) and lysates were also analysed by immunoblot with the indicated antibodies.
Figure Legend Snippet: Further evidence that SGK3 activity induced by inhibition of PI3K/Akt is regulated by hVps34 ZR‐75‐1 cells were treated with 1 μM MK‐2206 for 5 days, and then, 1 h prior to cell lysis, cells were further treated with increasing doses of SAR405. ZR‐75‐1 cells cultured in serum in the absence of Akt inhibitor were treated for 1 h with the indicated concentrations of SAR405. The cell lysates were analysed by immunoblot using the indicated antibodies. ZR‐75‐1 cells were treated for 5 days with 1 μM MK‐2206 or 1 μM GDC0941. One hour prior to lysis, the cells were incubated in the presence of absence of 0.3 μM SAR405. SGK3 was immunoprecipitated from lysates and subjected to in vitro kinase assay by measuring phosphorylation of the Crosstide substrate peptide in the presence of 0.1 mM [γ‐ 32 P]ATP in a 30 min 30°C reaction (upper panel). Kinase reactions are presented as means ± SD for triplicate reaction. Immunoprecipitates (IP) and lysates were also analysed by immunoblot with the indicated antibodies.

Techniques Used: Activity Assay, Inhibition, Lysis, Cell Culture, Incubation, Immunoprecipitation, In Vitro, Kinase Assay

Prolonged treatment with Class I PI 3K inhibitors leads to upregulation of SGK 3 The indicated cell lines were treated with either 1 μM MK‐2206, 1 μM AZD5363, 1 μM GDC0941 or 1 μM BKM120 for the indicated times. Cell lysates were subjected to immunoblot analysis with the indicated antibodies. The indicated cells were treated as in (A) and SGK3 was immunoprecipitated from the lysates using an anti‐SGK3 antibody. The immunoprecipitates (IP) were subjected to in vitro kinase assay by measuring phosphorylation of the Crosstide substrate peptide in the presence of 0.1 mM [γ‐ 32 P]ATP in a 30 min 30°C reaction (upper panel) followed by immunoblot analysis with the indicated antibodies (lower panel). Kinase reactions are presented as means ± SD for triplicate reaction. Source data are available online for this figure.
Figure Legend Snippet: Prolonged treatment with Class I PI 3K inhibitors leads to upregulation of SGK 3 The indicated cell lines were treated with either 1 μM MK‐2206, 1 μM AZD5363, 1 μM GDC0941 or 1 μM BKM120 for the indicated times. Cell lysates were subjected to immunoblot analysis with the indicated antibodies. The indicated cells were treated as in (A) and SGK3 was immunoprecipitated from the lysates using an anti‐SGK3 antibody. The immunoprecipitates (IP) were subjected to in vitro kinase assay by measuring phosphorylation of the Crosstide substrate peptide in the presence of 0.1 mM [γ‐ 32 P]ATP in a 30 min 30°C reaction (upper panel) followed by immunoblot analysis with the indicated antibodies (lower panel). Kinase reactions are presented as means ± SD for triplicate reaction. Source data are available online for this figure.

Techniques Used: Immunoprecipitation, In Vitro, Kinase Assay

14h selectively suppresses both the activity and activation of SGK 3 by PDK 1 and mTORC 2 Chemical structure of the Sanofi‐14h SGK inhibitor. IC 50 values of Sanofi‐14h SGK inhibitor on the indicated recombinant kinases. Protein kinase profiling undertaken against the Dundee panel of 140 protein kinases in the presence of 1 μM Sanofi‐14h at the International Centre for Protein Kinase Profiling. The result for each kinase is presented as a mean kinase activity of the reaction taken in triplicate relative to a control reaction where the inhibitors were omitted. Abbreviations and assay conditions are described at http://www.kinase-screen.mrc.ac.uk . ZR‐75‐1 cells were treated for 1 h with the indicated concentrations of 14h. The cell lysates were analysed by immunoblot analysis using the indicated antibodies. ZR‐75‐1 cells were treated for 1 h with the indicated concentrations of 14h. SGK3 was immunoprecipitated from cell lysates and subjected to in vitro kinase assay by measuring phosphorylation of the Crosstide substrate peptide in the presence of 0.1 mM [γ‐ 32 P]ATP in a 30 min 30°C reaction (upper panel). Kinase reactions are presented as means ± SD for triplicate reaction. Immunoprecipitates (IP) were also analysed by immunoblot with the indicated antibodies. The effect of the indicated concentration of 14h on the ability of SGK3[S486E]‐GST to be activated by PDK1 in the presence of PtdIns(3)P was assessed as described in Fig 4 .
Figure Legend Snippet: 14h selectively suppresses both the activity and activation of SGK 3 by PDK 1 and mTORC 2 Chemical structure of the Sanofi‐14h SGK inhibitor. IC 50 values of Sanofi‐14h SGK inhibitor on the indicated recombinant kinases. Protein kinase profiling undertaken against the Dundee panel of 140 protein kinases in the presence of 1 μM Sanofi‐14h at the International Centre for Protein Kinase Profiling. The result for each kinase is presented as a mean kinase activity of the reaction taken in triplicate relative to a control reaction where the inhibitors were omitted. Abbreviations and assay conditions are described at http://www.kinase-screen.mrc.ac.uk . ZR‐75‐1 cells were treated for 1 h with the indicated concentrations of 14h. The cell lysates were analysed by immunoblot analysis using the indicated antibodies. ZR‐75‐1 cells were treated for 1 h with the indicated concentrations of 14h. SGK3 was immunoprecipitated from cell lysates and subjected to in vitro kinase assay by measuring phosphorylation of the Crosstide substrate peptide in the presence of 0.1 mM [γ‐ 32 P]ATP in a 30 min 30°C reaction (upper panel). Kinase reactions are presented as means ± SD for triplicate reaction. Immunoprecipitates (IP) were also analysed by immunoblot with the indicated antibodies. The effect of the indicated concentration of 14h on the ability of SGK3[S486E]‐GST to be activated by PDK1 in the presence of PtdIns(3)P was assessed as described in Fig 4 .

Techniques Used: Activity Assay, Activation Assay, Recombinant, Immunoprecipitation, In Vitro, Kinase Assay, Concentration Assay

SGK 3 activity induced by inhibition of PI 3K/Akt is regulated by hV ps34 and mTORC 2 ZR‐75‐1 cells were treated with 1 μM MK‐2206 for 5 days and then, 1 h prior to cell lysis, cells were further treated with increasing doses of VPS34‐IN1. Cell lysates were subjected to immunoblot analysis with the indicated antibodies. ZR‐75‐1 cells cultured in serum in the absence of Akt inhibitor were treated for 1 h with the indicated concentrations of VPS34‐IN1. The cell lysates were analysed by immunoblot using the indicated antibodies. ZR‐75‐1 cells were treated for 5 days with either 1 μM MK‐2206, 1 μM AZD5363, 1 μM GDC0941 or 1 μM BKM120. One hour prior to lysis, the cells were incubated in the presence or absence of 1 μM VPS34‐IN1. SGK3 was immunoprecipitated from lysates and subjected to in vitro kinase assay by measuring phosphorylation of the Crosstide substrate peptide in the presence of 0.1 mM [γ‐ 32 P]ATP in a 30 min 30°C reaction (upper panel). Kinase reactions are presented as means ± SD for triplicate reaction. Immunoprecipitates (IP) and lysates were analysed by immunoblot with the indicated antibodies. One‐hour (1‐h) treatment with the PDK1 inhibitor GSK2334470 (Najafov et al , 2011 ) (1 μM) was used as a control for SGK3 inhibition. ZR‐75‐1 cells were stably transfected with either a control shRNA vector (scrambled) or a shRNA vector that targets Rictor expression (shRictor). The cells were grown in the presence or absence of 1 μM MK‐2206 or 1 μM GDC0941 for 5 days. SGK3 was immunoprecipitated from the lysates and subjected to in vitro kinase assay as in (C). Kinase reactions are presented as means ± SD for triplicate reaction. Immunoprecipitates (IP) and lysates (lower panel) were also subjected to immunoblot analysis with the indicated antibodies. ZR‐75‐1 cells were cultured in the absence or presence of 1 μM MK‐2206 for 5 days. Cells were then treated in the absence or presence of 0.1 μM AZD8055 or 0.1 μM rapamycin for 1 h. SGK3 was immunoprecipitated and subjected to in vitro kinase assay as in (C). Kinase reactions are presented as means ± SD for triplicate reaction. The immunoprecipitates (IP) and lysates were analysed with the indicated antibodies.
Figure Legend Snippet: SGK 3 activity induced by inhibition of PI 3K/Akt is regulated by hV ps34 and mTORC 2 ZR‐75‐1 cells were treated with 1 μM MK‐2206 for 5 days and then, 1 h prior to cell lysis, cells were further treated with increasing doses of VPS34‐IN1. Cell lysates were subjected to immunoblot analysis with the indicated antibodies. ZR‐75‐1 cells cultured in serum in the absence of Akt inhibitor were treated for 1 h with the indicated concentrations of VPS34‐IN1. The cell lysates were analysed by immunoblot using the indicated antibodies. ZR‐75‐1 cells were treated for 5 days with either 1 μM MK‐2206, 1 μM AZD5363, 1 μM GDC0941 or 1 μM BKM120. One hour prior to lysis, the cells were incubated in the presence or absence of 1 μM VPS34‐IN1. SGK3 was immunoprecipitated from lysates and subjected to in vitro kinase assay by measuring phosphorylation of the Crosstide substrate peptide in the presence of 0.1 mM [γ‐ 32 P]ATP in a 30 min 30°C reaction (upper panel). Kinase reactions are presented as means ± SD for triplicate reaction. Immunoprecipitates (IP) and lysates were analysed by immunoblot with the indicated antibodies. One‐hour (1‐h) treatment with the PDK1 inhibitor GSK2334470 (Najafov et al , 2011 ) (1 μM) was used as a control for SGK3 inhibition. ZR‐75‐1 cells were stably transfected with either a control shRNA vector (scrambled) or a shRNA vector that targets Rictor expression (shRictor). The cells were grown in the presence or absence of 1 μM MK‐2206 or 1 μM GDC0941 for 5 days. SGK3 was immunoprecipitated from the lysates and subjected to in vitro kinase assay as in (C). Kinase reactions are presented as means ± SD for triplicate reaction. Immunoprecipitates (IP) and lysates (lower panel) were also subjected to immunoblot analysis with the indicated antibodies. ZR‐75‐1 cells were cultured in the absence or presence of 1 μM MK‐2206 for 5 days. Cells were then treated in the absence or presence of 0.1 μM AZD8055 or 0.1 μM rapamycin for 1 h. SGK3 was immunoprecipitated and subjected to in vitro kinase assay as in (C). Kinase reactions are presented as means ± SD for triplicate reaction. The immunoprecipitates (IP) and lysates were analysed with the indicated antibodies.

Techniques Used: Activity Assay, Inhibition, Lysis, Cell Culture, Incubation, Immunoprecipitation, In Vitro, Kinase Assay, Stable Transfection, Transfection, shRNA, Plasmid Preparation, Expressing

36) Product Images from "Kaempferol Downregulates Insulin-like Growth Factor-I Receptor and ErbB3 Signaling in HT-29 Human Colon Cancer Cells"

Article Title: Kaempferol Downregulates Insulin-like Growth Factor-I Receptor and ErbB3 Signaling in HT-29 Human Colon Cancer Cells

Journal: Journal of Cancer Prevention

doi: 10.15430/JCP.2014.19.2.161

Effect of kaempferol on the insulin-like growth factor-I receptor (IGF-IR) signaling pathway. HT-29 cells were treated with 0 or 60 μmol/L kaempferol for 2 hours and lysed after 0, 1, or 5 minutes of stimulation with 10 nmol/L insulin-like growth factor-I (IGF-I). (A) Total cell lysates were incubated with anti-IGF-IRβ antibody and the immune complexes were precipitated with protein A-Sepharose. The immunoprecipitated proteins were subjected to Western blot analysis with the relevant antibodies. The immunoprecipitated proteins were incubated with phosphatidylinositol and [γ- 32 P]ATP to detect phosphatidylinositol 3-kinase (PI3K) activity. The resulting 32 P-labelled phosphatidylinositol 3-phosphate (PIP) was separated by thin-layer chromatography and visualized by autoradiography. (B) Total cell lysates were subjected to Western blot analysis with their relevant antibodies. ERK, extracellular regulated kinase; IP, immunoprecipitation; WB, western blot analysis.
Figure Legend Snippet: Effect of kaempferol on the insulin-like growth factor-I receptor (IGF-IR) signaling pathway. HT-29 cells were treated with 0 or 60 μmol/L kaempferol for 2 hours and lysed after 0, 1, or 5 minutes of stimulation with 10 nmol/L insulin-like growth factor-I (IGF-I). (A) Total cell lysates were incubated with anti-IGF-IRβ antibody and the immune complexes were precipitated with protein A-Sepharose. The immunoprecipitated proteins were subjected to Western blot analysis with the relevant antibodies. The immunoprecipitated proteins were incubated with phosphatidylinositol and [γ- 32 P]ATP to detect phosphatidylinositol 3-kinase (PI3K) activity. The resulting 32 P-labelled phosphatidylinositol 3-phosphate (PIP) was separated by thin-layer chromatography and visualized by autoradiography. (B) Total cell lysates were subjected to Western blot analysis with their relevant antibodies. ERK, extracellular regulated kinase; IP, immunoprecipitation; WB, western blot analysis.

Techniques Used: Incubation, Immunoprecipitation, Western Blot, Activity Assay, Thin Layer Chromatography, Autoradiography

37) Product Images from "Phosphate Sink Containing Two-Component Signaling Systems as Tunable Threshold Devices"

Article Title: Phosphate Sink Containing Two-Component Signaling Systems as Tunable Threshold Devices

Journal: PLoS Computational Biology

doi: 10.1371/journal.pcbi.1003890

Experimental validation for the role of the sink RR in shaping the signal-response curve. The steady-state level of phosphorylated CheY2 was measured in the presence or absence of the sink (i.e. CheY1) at different 32 P-ATP concentrations. ( A ) Phosphorimages showing phosphorylated CheY2 levels in the presence or absence of CheY1 at low (0.2 mM) and high (2 mM) ATP levels. The indicated quantity of [γ- 32 P] ATP was added to a reaction mixture containing 10 µM CheA, 2.5 µM CheY2, and where indicated 2.5 µM CheY1. ( B ) Graph comparing the observed steady state levels of phosphorylated CheY2 with and without the sink, CheY1. The phosphorylated CheY2 levels predicted by the model are shown with a dashed line (in absence of sink) and with a solid line (in presence of sink), while the experimentally measured values are shown by squares (in absence of sink) and circles (in presence of sink). Error bars show the standard error of the mean obtained from three independent experiments.
Figure Legend Snippet: Experimental validation for the role of the sink RR in shaping the signal-response curve. The steady-state level of phosphorylated CheY2 was measured in the presence or absence of the sink (i.e. CheY1) at different 32 P-ATP concentrations. ( A ) Phosphorimages showing phosphorylated CheY2 levels in the presence or absence of CheY1 at low (0.2 mM) and high (2 mM) ATP levels. The indicated quantity of [γ- 32 P] ATP was added to a reaction mixture containing 10 µM CheA, 2.5 µM CheY2, and where indicated 2.5 µM CheY1. ( B ) Graph comparing the observed steady state levels of phosphorylated CheY2 with and without the sink, CheY1. The phosphorylated CheY2 levels predicted by the model are shown with a dashed line (in absence of sink) and with a solid line (in presence of sink), while the experimentally measured values are shown by squares (in absence of sink) and circles (in presence of sink). Error bars show the standard error of the mean obtained from three independent experiments.

Techniques Used:

38) Product Images from "Stress-Stimulated Mitogen-Activated Protein Kinases Control the Stability and Activity of the Cdt1 DNA Replication Licensing Factor ▿"

Article Title: Stress-Stimulated Mitogen-Activated Protein Kinases Control the Stability and Activity of the Cdt1 DNA Replication Licensing Factor ▿

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.06163-11

Five sites in Cdt1 are phosphorylated by stress-inducible MAP kinases. (A) Recombinant full-length N-terminal GST-Cdt1 fusion proteins were incubated with active p38 in the presence of [γ- 32 P]ATP followed by autoradiography. Total Cdt1 was detected
Figure Legend Snippet: Five sites in Cdt1 are phosphorylated by stress-inducible MAP kinases. (A) Recombinant full-length N-terminal GST-Cdt1 fusion proteins were incubated with active p38 in the presence of [γ- 32 P]ATP followed by autoradiography. Total Cdt1 was detected

Techniques Used: Recombinant, Incubation, Autoradiography

39) Product Images from "A GAPDH Mutant Defective in Src-Dependent Tyrosine Phosphorylation Impedes Rab2-Mediated Events"

Article Title: A GAPDH Mutant Defective in Src-Dependent Tyrosine Phosphorylation Impedes Rab2-Mediated Events

Journal: Traffic (Copenhagen, Denmark)

doi: 10.1111/j.1600-0854.2007.00569.x

Src does not Phosphorylate GAPDH Y41F Src (0.1 µg) was incubated with purified recombinant GAPDH or GAPDH Y41F (0.5 µg) in 50 mM Tris (pH 7.4), 10 mM MgCl 2 , 3 mM MnCl 2 , 50 µM ATP, and 10 µCi [γ- 32 P]ATP for 15 min at 30°C in the absence or presence of 5 µM PP2. The reaction was stopped by the addition of 5X sample buffer, and then separated by SDS-PAGE followed by autoradiography.
Figure Legend Snippet: Src does not Phosphorylate GAPDH Y41F Src (0.1 µg) was incubated with purified recombinant GAPDH or GAPDH Y41F (0.5 µg) in 50 mM Tris (pH 7.4), 10 mM MgCl 2 , 3 mM MnCl 2 , 50 µM ATP, and 10 µCi [γ- 32 P]ATP for 15 min at 30°C in the absence or presence of 5 µM PP2. The reaction was stopped by the addition of 5X sample buffer, and then separated by SDS-PAGE followed by autoradiography.

Techniques Used: Incubation, Purification, Recombinant, SDS Page, Autoradiography

40) Product Images from "The predicted truncation from a cancer-associated variant of the MSH2 initiation codon alters activity of the MSH2-MSH6 mismatch repair complex"

Article Title: The predicted truncation from a cancer-associated variant of the MSH2 initiation codon alters activity of the MSH2-MSH6 mismatch repair complex

Journal: Molecular Carcinogenesis

doi: 10.1002/mc.20838

Nucleotide binding and exchange properties of MSH2(NΔ25)-MSH6. (A) ATP binding activity. Wild-type MSH2-MSH6 and MSH2(NΔ25)-MSH6 proteins were UV- crosslinked to radiolabeled [γ- 32 P]ATP in the presence or absence of Mg 2+ (to allow
Figure Legend Snippet: Nucleotide binding and exchange properties of MSH2(NΔ25)-MSH6. (A) ATP binding activity. Wild-type MSH2-MSH6 and MSH2(NΔ25)-MSH6 proteins were UV- crosslinked to radiolabeled [γ- 32 P]ATP in the presence or absence of Mg 2+ (to allow

Techniques Used: Binding Assay, Activity Assay

41) Product Images from "Two Pdk1 phosphorylation sites on the plant cell death suppressor Adi3 contribute to substrate phosphorylation"

Article Title: Two Pdk1 phosphorylation sites on the plant cell death suppressor Adi3 contribute to substrate phosphorylation

Journal: Biochimica et biophysica acta

doi: 10.1016/j.bbapap.2013.03.006

Activation of Adi3 kinase activity toward Gal83 through Ser212 and Ser539 phosphorylation. In A and B, the indicated proteins were incubated in an in vitro kinase assay with γ-[ 32 P]ATP. Top panel, phosphorimage; bottom panel, Coomassie stained
Figure Legend Snippet: Activation of Adi3 kinase activity toward Gal83 through Ser212 and Ser539 phosphorylation. In A and B, the indicated proteins were incubated in an in vitro kinase assay with γ-[ 32 P]ATP. Top panel, phosphorimage; bottom panel, Coomassie stained

Techniques Used: Activation Assay, Activity Assay, Incubation, In Vitro, Kinase Assay, Staining

Contribution of MS/MS-identified Adi3 phosphorylation sites to phosphorylation by Sl Pdk1 and Adi3 trans phosphorylation. In (A) and (B), the indicated proteins were incubated in an in vitro kinase assay with γ-[ 32 P]ATP. Top panel, phosphorimage;
Figure Legend Snippet: Contribution of MS/MS-identified Adi3 phosphorylation sites to phosphorylation by Sl Pdk1 and Adi3 trans phosphorylation. In (A) and (B), the indicated proteins were incubated in an in vitro kinase assay with γ-[ 32 P]ATP. Top panel, phosphorimage;

Techniques Used: Mass Spectrometry, Incubation, In Vitro, Kinase Assay

Identification of Ser269 as a second phosphorylation site in AGC1-3. In (A) and (C), the indicated proteins were incubated in an in vitro kinase assay with γ-[ 32 P]ATP. Top panel, phosphorimage; bottom panel, Coomassie stained gel; middle panel,
Figure Legend Snippet: Identification of Ser269 as a second phosphorylation site in AGC1-3. In (A) and (C), the indicated proteins were incubated in an in vitro kinase assay with γ-[ 32 P]ATP. Top panel, phosphorimage; bottom panel, Coomassie stained gel; middle panel,

Techniques Used: Incubation, In Vitro, Kinase Assay, Staining

Evidence for Adi3 Ser212 phosphorylation by Sl Pdk1. In A and B, the indicated proteins were incubated in an in vitro kinase assay with γ-[ 32 P]ATP. Top panel, phosphorimage; bottom panel, Coomassie stained gel; middle panel, quantification of Adi3
Figure Legend Snippet: Evidence for Adi3 Ser212 phosphorylation by Sl Pdk1. In A and B, the indicated proteins were incubated in an in vitro kinase assay with γ-[ 32 P]ATP. Top panel, phosphorimage; bottom panel, Coomassie stained gel; middle panel, quantification of Adi3

Techniques Used: Incubation, In Vitro, Kinase Assay, Staining

42) 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

43) Product Images from "Unencumbered Pol β lyase activity in nucleosome core particles"

Article Title: Unencumbered Pol β lyase activity in nucleosome core particles

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkx593

Pol β dRP lyase activity assay. ( A ) DNA oligo (15-mer) was 5′-end labeled with γ- 32 P-ATP. Experiments were conducted as described in Materials and Methods. The reaction with Pol β was initiated ∼30 s after UDG incubation. A negative control was included to subtract the spontaneous loss of the 5′-dRP. A heat control (H), as described in Materials and Methods, was included to determine the actual dRP substrate generated after the 5.5-min UDG incubation. Full length Pol β (FL) or the N-terminal domain of Pol β (8 kDa) were used to determine dRP lyase activity. A representative phosphor image of a 20% polyacrylamide denaturing gel of the replicates plotted in panel (B) is shown. ( B ) NCP crystal structure pdb file 3LZ0 ( 37 ) was modified to show the structural location of the 5′-dRP group (black spheres). This is the back view of that shown in Figure 1 . Data represent the mean of three independent experiments ± SD. Kinetic parameters are provided in Table 2 (full length Pol β) and Table 3 (8 kDa domain of Pol β). As shown in Table 2 , the ratio (DNA/NCP) of the enzymatic rates for this 5′-end labeled substrate is comparable to the ratio found by the 3′-end labeling assay ( Supplementary Figure S3 ). The rate of spontaneous loss, estimated from the –Pol β control, for the NCP is ∼3.2 nM/min, which is 8-fold faster than the rate in DNA of 0.4 nM/min.
Figure Legend Snippet: Pol β dRP lyase activity assay. ( A ) DNA oligo (15-mer) was 5′-end labeled with γ- 32 P-ATP. Experiments were conducted as described in Materials and Methods. The reaction with Pol β was initiated ∼30 s after UDG incubation. A negative control was included to subtract the spontaneous loss of the 5′-dRP. A heat control (H), as described in Materials and Methods, was included to determine the actual dRP substrate generated after the 5.5-min UDG incubation. Full length Pol β (FL) or the N-terminal domain of Pol β (8 kDa) were used to determine dRP lyase activity. A representative phosphor image of a 20% polyacrylamide denaturing gel of the replicates plotted in panel (B) is shown. ( B ) NCP crystal structure pdb file 3LZ0 ( 37 ) was modified to show the structural location of the 5′-dRP group (black spheres). This is the back view of that shown in Figure 1 . Data represent the mean of three independent experiments ± SD. Kinetic parameters are provided in Table 2 (full length Pol β) and Table 3 (8 kDa domain of Pol β). As shown in Table 2 , the ratio (DNA/NCP) of the enzymatic rates for this 5′-end labeled substrate is comparable to the ratio found by the 3′-end labeling assay ( Supplementary Figure S3 ). The rate of spontaneous loss, estimated from the –Pol β control, for the NCP is ∼3.2 nM/min, which is 8-fold faster than the rate in DNA of 0.4 nM/min.

Techniques Used: Activity Assay, Labeling, Incubation, Negative Control, Generated, Modification, End Labeling

44) Product Images from "Molecular mechanisms of two-component system RhpRS regulating type III secretion system in Pseudomonas syringae"

Article Title: Molecular mechanisms of two-component system RhpRS regulating type III secretion system in Pseudomonas syringae

Journal: Nucleic Acids Research

doi: 10.1093/nar/gku865

Kinase activity of RhpS C and DNA binding activity of RhpR. ( A ) RhpS C (2 μM) was mixed with [γ- 32 P]-ATP for 30 min before adding RhpR (10 μM) into the reaction mixture for 120 min. Phosphorylated RhpS C was not able to phosphorylate non-cognate GacA over a 30 min period. ( B ) DNA binding activities of unphosphorylated RhpR (left panel) and phosphorylated RhpR (P-RhpR, right panel) to its own promoter are shown. RhpR was pre-mixed with RhpS C in the presence or absence of ATP. Aliquots containing the indicated amount of RhpR protein were mixed with 2 ng of a γ- 32 P-end-labeled rhpR promoter fragment in EMSA buffer at room temperature for 30 min before performing a gel shift assay. ( C ) RhpR does not bind to the promoter region of PSPTO_1489.
Figure Legend Snippet: Kinase activity of RhpS C and DNA binding activity of RhpR. ( A ) RhpS C (2 μM) was mixed with [γ- 32 P]-ATP for 30 min before adding RhpR (10 μM) into the reaction mixture for 120 min. Phosphorylated RhpS C was not able to phosphorylate non-cognate GacA over a 30 min period. ( B ) DNA binding activities of unphosphorylated RhpR (left panel) and phosphorylated RhpR (P-RhpR, right panel) to its own promoter are shown. RhpR was pre-mixed with RhpS C in the presence or absence of ATP. Aliquots containing the indicated amount of RhpR protein were mixed with 2 ng of a γ- 32 P-end-labeled rhpR promoter fragment in EMSA buffer at room temperature for 30 min before performing a gel shift assay. ( C ) RhpR does not bind to the promoter region of PSPTO_1489.

Techniques Used: Activity Assay, Binding Assay, Labeling, Electrophoretic Mobility Shift Assay

45) Product Images from "Pathways Leading from BarA/SirA to Motility and Virulence Gene Expression in Salmonella"

Article Title: Pathways Leading from BarA/SirA to Motility and Virulence Gene Expression in Salmonella

Journal: Journal of Bacteriology

doi: 10.1128/JB.185.24.7257-7265.2003

Purified BarA198 can autophosphorylate and transfer phosphate to SirA in vitro. (A) Purified SirA and BarA198 after nickel affinity chromatography, SDS-PAGE, and Coomassie blue staining. Both proteins are His 6 tagged. Molecular mass markers (Bio-Rad prestained broad-range standards) are in the center lane (from bottom to top: 7.1, 21, 29, 35, 49, 80, 124, and 209 kDa). SirA-His 6 is predicted to be 24.95 kDa; His 6 -BarA198 is predicted to be 81.60 kDa. (B) Time course of BarA198 autophosphorylation in the presence of [γ- 32 P]ATP. Reactions were allowed to proceed for the times indicated above each lane in minutes before aliquots were removed and the reactions were stopped by the addition of sample buffer containing SDS. Samples were resolved by SDS-PAGE and detected with a PhosphorImager. (C) Plot of band densities as determined by ImageQuant software from panel B. (D) Transphosphorylation of SirA by BarA198-P. BarA198 that had been preincubated with [γ- 32 P]ATP for 25 min was mixed with SirA as indicated. One reaction contained SirA with [γ- 32 P]ATP and no BarA198. Reactions were stopped at the times indicated above each lane in minutes by the addition of sample buffer containing SDS. Samples were resolved by SDS-PAGE and detected with a PhosphorImager. (E) Plot of band densities as determined by ImageQuant software from panel D. P, phosphorylated.
Figure Legend Snippet: Purified BarA198 can autophosphorylate and transfer phosphate to SirA in vitro. (A) Purified SirA and BarA198 after nickel affinity chromatography, SDS-PAGE, and Coomassie blue staining. Both proteins are His 6 tagged. Molecular mass markers (Bio-Rad prestained broad-range standards) are in the center lane (from bottom to top: 7.1, 21, 29, 35, 49, 80, 124, and 209 kDa). SirA-His 6 is predicted to be 24.95 kDa; His 6 -BarA198 is predicted to be 81.60 kDa. (B) Time course of BarA198 autophosphorylation in the presence of [γ- 32 P]ATP. Reactions were allowed to proceed for the times indicated above each lane in minutes before aliquots were removed and the reactions were stopped by the addition of sample buffer containing SDS. Samples were resolved by SDS-PAGE and detected with a PhosphorImager. (C) Plot of band densities as determined by ImageQuant software from panel B. (D) Transphosphorylation of SirA by BarA198-P. BarA198 that had been preincubated with [γ- 32 P]ATP for 25 min was mixed with SirA as indicated. One reaction contained SirA with [γ- 32 P]ATP and no BarA198. Reactions were stopped at the times indicated above each lane in minutes by the addition of sample buffer containing SDS. Samples were resolved by SDS-PAGE and detected with a PhosphorImager. (E) Plot of band densities as determined by ImageQuant software from panel D. P, phosphorylated.

Techniques Used: Purification, In Vitro, Affinity Chromatography, SDS Page, Staining, Software

SirA alters the gel mobilities of promoter DNA fragments. Eight promoter DNA fragments (shown below panels) were tested for their abilities to bind purified SirA in a gel mobility shift assay. SirA was phosphorylated by incubation with BarA198 and ATP for 25 min and then was added to promoter DNA labeled with [γ- 32 P]ATP. Each reaction was resolved by nondenaturing PAGE, and samples were detected with a PhosphorImager. The micromolar concentration of SirA in each reaction is indicated above each lane. An asterisk indicates that a 30- to 50-fold excess of unlabeled promoter DNA fragment was added to the reaction as a specific competitor. All reactions contained nonspecific competitor DNA [2 μg of poly(dI-dC)] and protein (0.2 μg of BSA).
Figure Legend Snippet: SirA alters the gel mobilities of promoter DNA fragments. Eight promoter DNA fragments (shown below panels) were tested for their abilities to bind purified SirA in a gel mobility shift assay. SirA was phosphorylated by incubation with BarA198 and ATP for 25 min and then was added to promoter DNA labeled with [γ- 32 P]ATP. Each reaction was resolved by nondenaturing PAGE, and samples were detected with a PhosphorImager. The micromolar concentration of SirA in each reaction is indicated above each lane. An asterisk indicates that a 30- to 50-fold excess of unlabeled promoter DNA fragment was added to the reaction as a specific competitor. All reactions contained nonspecific competitor DNA [2 μg of poly(dI-dC)] and protein (0.2 μg of BSA).

Techniques Used: Purification, Mobility Shift, Incubation, Labeling, Polyacrylamide Gel Electrophoresis, Concentration Assay

46) Product Images from ""

Article Title:

Journal: Molecular Pharmacology

doi: 10.1124/mol.113.090837

Leucettine L41 potentiates CLK1 siRNA-induced autophagy through PIKfyve inhibition. (A) L41 treatment induces vacuole formation. (Left) Differential interference contrast picture of untreated U-2 OS cells (Cntrl) or treated for 24 hours with 20 μ M L41 (L41) or with PIKfyve siRNA for 68 hours. Arrowheads point to vacuoles. (Right) Quantification of phenotype observed on the left panels. (B) Inhibition of PIKfyve triggers autophagy. Quantification of LC3 foci in cells treated with DMSO (Cntrl), rapamycin or the PIKfyve inhibitor YM201636. (C) Inhibition of recombinant PIKfyve activity by increasing doses of L41 (red line, open circles) and YM201636 (black line, black squares). The PIKfyve assay was performed in the presence of its substrate PtdIns3P and [γ- 32 P]ATP. The lipid substrate and product were separated by thin-layer chromatography. The amount of 32 P-PtdIns(3,5)P 2 produced was quantified and the results are expressed as percentage of maximal, uninhibited activity, and are mean of two to three independent experiments. (D) Schematic simplified representation of the phosphoinositide metabolism. Note that PIKfyve catalyzes the conversion of PtdIns(3)P to PtdIns(3,5)P 2 and in certain conditions PtdIns to PtdIns5P. (E) Effect of various doses of L41 on the steady-state levels of the phosphoinositides, as measured in vivo in myo-[2- 3 H]inositol labeled differentiated 3T3L1 adipocytes by HPLC analyses. Individual peak radioactivity was quantified by area integration and presented as a percentage of the summed radioactivity from the [ 3 H]GroPIns3P, -4P, -5P, -(3,5)P 2 , and -(4,5)P 2 peaks (“total radioactivity”); mean of three independent experiments. (F, left) Cells were subjected to control (Cntrl), CLK1, PIKfyve siRNA, and L41 (co)treatment as indicated. The number of LC3 foci was scored. (Right) Immunofluorescence images showing representative examples of the phenotype quantified on the leftmost panel. DNA is stained with 4′,6-diamidino-2-phenylindole (blue), LC3 is in green. (Means of at least three independent experiments; more than 150 cells were analyzed per experiment; error bars show S.E.M.; * P
Figure Legend Snippet: Leucettine L41 potentiates CLK1 siRNA-induced autophagy through PIKfyve inhibition. (A) L41 treatment induces vacuole formation. (Left) Differential interference contrast picture of untreated U-2 OS cells (Cntrl) or treated for 24 hours with 20 μ M L41 (L41) or with PIKfyve siRNA for 68 hours. Arrowheads point to vacuoles. (Right) Quantification of phenotype observed on the left panels. (B) Inhibition of PIKfyve triggers autophagy. Quantification of LC3 foci in cells treated with DMSO (Cntrl), rapamycin or the PIKfyve inhibitor YM201636. (C) Inhibition of recombinant PIKfyve activity by increasing doses of L41 (red line, open circles) and YM201636 (black line, black squares). The PIKfyve assay was performed in the presence of its substrate PtdIns3P and [γ- 32 P]ATP. The lipid substrate and product were separated by thin-layer chromatography. The amount of 32 P-PtdIns(3,5)P 2 produced was quantified and the results are expressed as percentage of maximal, uninhibited activity, and are mean of two to three independent experiments. (D) Schematic simplified representation of the phosphoinositide metabolism. Note that PIKfyve catalyzes the conversion of PtdIns(3)P to PtdIns(3,5)P 2 and in certain conditions PtdIns to PtdIns5P. (E) Effect of various doses of L41 on the steady-state levels of the phosphoinositides, as measured in vivo in myo-[2- 3 H]inositol labeled differentiated 3T3L1 adipocytes by HPLC analyses. Individual peak radioactivity was quantified by area integration and presented as a percentage of the summed radioactivity from the [ 3 H]GroPIns3P, -4P, -5P, -(3,5)P 2 , and -(4,5)P 2 peaks (“total radioactivity”); mean of three independent experiments. (F, left) Cells were subjected to control (Cntrl), CLK1, PIKfyve siRNA, and L41 (co)treatment as indicated. The number of LC3 foci was scored. (Right) Immunofluorescence images showing representative examples of the phenotype quantified on the leftmost panel. DNA is stained with 4′,6-diamidino-2-phenylindole (blue), LC3 is in green. (Means of at least three independent experiments; more than 150 cells were analyzed per experiment; error bars show S.E.M.; * P

Techniques Used: Inhibition, Recombinant, Activity Assay, Thin Layer Chromatography, Produced, In Vivo, Labeling, High Performance Liquid Chromatography, Radioactivity, Immunofluorescence, Staining

47) Product Images from "Cyclin-Dependent Kinase 5 Phosphorylation of Human Septin SEPT5 (hCDCrel-1) Modulates Exocytosis"

Article Title: Cyclin-Dependent Kinase 5 Phosphorylation of Human Septin SEPT5 (hCDCrel-1) Modulates Exocytosis

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.0453-08.2008

Serine 327 is the principal site on SEPT5 that is phosphorylated by Cdk5/p35. GST-SEPT5 was phosphorylated in vitro by Cdk5/p35 with [γ- 32 P]ATP, subjected to SDS-PAGE, and transferred to a PVDF membrane. The radioactive band was removed and digested with trypsin. A ) in which sequence cycles are collected as spots on filter papers in a microtiter plate and assayed for radioactivity. B , In the third cycle, the 32 P signal is derived from a serine 3 residue from the N-terminal end of the tryptic peptide. The insert sequence shows the tryptic digestion site (arrow), and note that the phosphorylated S (*) at the third residue from the N-terminal end is S327. C , At the ninth cycle where activity is significantly lower, the 32 P signal marks a serine 9 residue from the N-terminal end of the tryptic peptide. The sequence in the insert, representing the only SEPT5 sequence satisfying this criterion, is shown with the tryptic digest site at the arrow, and the S* (S161), the ninth residue from the N-terminal end of the peptide.
Figure Legend Snippet: Serine 327 is the principal site on SEPT5 that is phosphorylated by Cdk5/p35. GST-SEPT5 was phosphorylated in vitro by Cdk5/p35 with [γ- 32 P]ATP, subjected to SDS-PAGE, and transferred to a PVDF membrane. The radioactive band was removed and digested with trypsin. A ) in which sequence cycles are collected as spots on filter papers in a microtiter plate and assayed for radioactivity. B , In the third cycle, the 32 P signal is derived from a serine 3 residue from the N-terminal end of the tryptic peptide. The insert sequence shows the tryptic digestion site (arrow), and note that the phosphorylated S (*) at the third residue from the N-terminal end is S327. C , At the ninth cycle where activity is significantly lower, the 32 P signal marks a serine 9 residue from the N-terminal end of the tryptic peptide. The sequence in the insert, representing the only SEPT5 sequence satisfying this criterion, is shown with the tryptic digest site at the arrow, and the S* (S161), the ninth residue from the N-terminal end of the peptide.

Techniques Used: In Vitro, SDS Page, Sequencing, Radioactivity, Derivative Assay, Activity Assay

48) Product Images from "Extent to which hairpin opening by the Artemis:DNA-PKcs complex can contribute to junctional diversity in V(D)J recombination"

Article Title: Extent to which hairpin opening by the Artemis:DNA-PKcs complex can contribute to junctional diversity in V(D)J recombination

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkm823

The phosphorylation of Artemis by DNA-PKcs rapidly reaches a plateau. ( A ) Artemis was incubated with DNA-PKcs in the presence of [γ- 32 P] ATP at 37°C. A fraction of the incubation mixture was removed at each time point and resolved on SDS–PAGE. For the zero time point, a separate reaction containing all components except DNA-PKcs was used. ( B ) The level of phosphorylation was quantified and normalized as a percentage of the highest level (100% at 30 min).
Figure Legend Snippet: The phosphorylation of Artemis by DNA-PKcs rapidly reaches a plateau. ( A ) Artemis was incubated with DNA-PKcs in the presence of [γ- 32 P] ATP at 37°C. A fraction of the incubation mixture was removed at each time point and resolved on SDS–PAGE. For the zero time point, a separate reaction containing all components except DNA-PKcs was used. ( B ) The level of phosphorylation was quantified and normalized as a percentage of the highest level (100% at 30 min).

Techniques Used: Incubation, SDS Page

49) Product Images from "Metabolism of circulating ADP in the bloodstream is mediated via integrated actions of soluble adenylate kinase-1 and NTPDase1/CD39 activities"

Article Title: Metabolism of circulating ADP in the bloodstream is mediated via integrated actions of soluble adenylate kinase-1 and NTPDase1/CD39 activities

Journal: The FASEB Journal

doi: 10.1096/fj.12-205658

TLC analysis of purine-converting pathways in the serum from AK1 −/− mice. A , B ) Serum from AK1 −/− and wild-type (AK +/+ ) mice was incubated with 50 μM [γ- 32 P]ATP in the absence (vehicle) or presence of 1
Figure Legend Snippet: TLC analysis of purine-converting pathways in the serum from AK1 −/− mice. A , B ) Serum from AK1 −/− and wild-type (AK +/+ ) mice was incubated with 50 μM [γ- 32 P]ATP in the absence (vehicle) or presence of 1

Techniques Used: Thin Layer Chromatography, Mouse Assay, Incubation

TLC analysis of soluble nucleotidase activities in CD39 −/− mice. A , B ) Sera from CD39 −/− and wild-type (CD39 +/+ ) mice were incubated with 50 μM [γ- 32 P]ATP ( A ) or with 100 μM [ 3 H]ADP ( B ). Some samples
Figure Legend Snippet: TLC analysis of soluble nucleotidase activities in CD39 −/− mice. A , B ) Sera from CD39 −/− and wild-type (CD39 +/+ ) mice were incubated with 50 μM [γ- 32 P]ATP ( A ) or with 100 μM [ 3 H]ADP ( B ). Some samples

Techniques Used: Thin Layer Chromatography, Mouse Assay, Incubation

50) Product Images from "Algal Dual-Specificity Tyrosine Phosphorylation-Regulated Kinase, Triacylglycerol Accumulation Regulator1, Regulates Accumulation of Triacylglycerol in Nitrogen or Sulfur Deficiency 1Algal Dual-Specificity Tyrosine Phosphorylation-Regulated Kinase, Triacylglycerol Accumulation Regulator1, Regulates Accumulation of Triacylglycerol in Nitrogen or Sulfur Deficiency 1 [OPEN]"

Article Title: Algal Dual-Specificity Tyrosine Phosphorylation-Regulated Kinase, Triacylglycerol Accumulation Regulator1, Regulates Accumulation of Triacylglycerol in Nitrogen or Sulfur Deficiency 1Algal Dual-Specificity Tyrosine Phosphorylation-Regulated Kinase, Triacylglycerol Accumulation Regulator1, Regulates Accumulation of Triacylglycerol in Nitrogen or Sulfur Deficiency 1 [OPEN]

Journal: Plant Physiology

doi: 10.1104/pp.15.00319

In vitro kinase assay using pTAR1. A pTAR1-containing kinase domain and that of a K523R substitution of the putative phosphate anchor Lys (pTAR1 KD ) were incubated with [γ- 32 P]ATP in the presence or absence of Mg 2+ . Proteins are stained with Coomassie
Figure Legend Snippet: In vitro kinase assay using pTAR1. A pTAR1-containing kinase domain and that of a K523R substitution of the putative phosphate anchor Lys (pTAR1 KD ) were incubated with [γ- 32 P]ATP in the presence or absence of Mg 2+ . Proteins are stained with Coomassie

Techniques Used: In Vitro, Kinase Assay, Incubation, Staining

51) Product Images from "Intrinsic Negative Feedback Governs Activation Surge in Two-Component Regulatory Systems"

Article Title: Intrinsic Negative Feedback Governs Activation Surge in Two-Component Regulatory Systems

Journal: Molecular cell

doi: 10.1016/j.molcel.2011.12.027

Purified PhoQ and PhoP Proteins Recapitulate the PhoP-P Surge in the Presence of ATP and ADP In Vitro (A) Levels of PhoP-P and PhoQ-P at the indicated times after incubation of PhoP with wild-type PhoQ protein in the presence of ATP (1 mM) and ADP (0.1 mM). (B) Quantitation of the in vitro surge assay shown in (A). The graph depicts the absolute amount of PhoP-P (open circles) and PhoQ-P (closed circles). Inset: The normalized levels of PhoP-P (open circles) and PhoQ-P (closed circles) relative to their maximum levels. Data correspond to the average ± SEM from three independent experiments. (C) ATP remaining in the reaction after incubation with PhoP and PhoQ proteins. ATP and Pi were visualized following a run on PEI-cellulose TLC plate. [γ- 32 P] ATP was used as a marker. The positions of ATP, Pi, PhoP-P, and PhoQ-P are indicated by arrows. (D) Levels of PhoP and PhoQ proteins during in vitro surge were visualized on SDS-PAGE. The positions of PhoP and PhoQ are marked with arrows and a standard protein size marker was run in parallel. .
Figure Legend Snippet: Purified PhoQ and PhoP Proteins Recapitulate the PhoP-P Surge in the Presence of ATP and ADP In Vitro (A) Levels of PhoP-P and PhoQ-P at the indicated times after incubation of PhoP with wild-type PhoQ protein in the presence of ATP (1 mM) and ADP (0.1 mM). (B) Quantitation of the in vitro surge assay shown in (A). The graph depicts the absolute amount of PhoP-P (open circles) and PhoQ-P (closed circles). Inset: The normalized levels of PhoP-P (open circles) and PhoQ-P (closed circles) relative to their maximum levels. Data correspond to the average ± SEM from three independent experiments. (C) ATP remaining in the reaction after incubation with PhoP and PhoQ proteins. ATP and Pi were visualized following a run on PEI-cellulose TLC plate. [γ- 32 P] ATP was used as a marker. The positions of ATP, Pi, PhoP-P, and PhoQ-P are indicated by arrows. (D) Levels of PhoP and PhoQ proteins during in vitro surge were visualized on SDS-PAGE. The positions of PhoP and PhoQ are marked with arrows and a standard protein size marker was run in parallel. .

Techniques Used: Purification, In Vitro, Incubation, Quantitation Assay, Thin Layer Chromatography, Marker, SDS Page

52) Product Images from "Phosphoryl Group Flow within the Pseudomonas aeruginosa Pil-Chp Chemosensory System: DIFFERENTIAL FUNCTION OF THE EIGHT PHOSPHOTRANSFERASE AND THREE RECEIVER DOMAINS*"

Article Title: Phosphoryl Group Flow within the Pseudomonas aeruginosa Pil-Chp Chemosensory System: DIFFERENTIAL FUNCTION OF THE EIGHT PHOSPHOTRANSFERASE AND THREE RECEIVER DOMAINS*

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M116.737528

Time courses for autophosphorylation of ChpA constructs that do not contain the C-terminal receiver domain (ChpArec). The ChpA construct (1.4 μ m ) was incubated with [γ- 32 P]ATP (150 μ m ) in 50 m m Tris, pH 7.5, 50 m m KCl, 5 m m MgCl
Figure Legend Snippet: Time courses for autophosphorylation of ChpA constructs that do not contain the C-terminal receiver domain (ChpArec). The ChpA construct (1.4 μ m ) was incubated with [γ- 32 P]ATP (150 μ m ) in 50 m m Tris, pH 7.5, 50 m m KCl, 5 m m MgCl

Techniques Used: Construct, Incubation

Can Hpt2, Hpt3, and Hpt4 transfer rapidly to ChpArec, PilG, or PilH? Gel phosphorimages following phosphotransfer from Xpts (Hpt2–4) to PilG, PilH, and ChpArec. Reactions were initiated by addition of 30 μ m [γ- 32 P]ATP to mixtures
Figure Legend Snippet: Can Hpt2, Hpt3, and Hpt4 transfer rapidly to ChpArec, PilG, or PilH? Gel phosphorimages following phosphotransfer from Xpts (Hpt2–4) to PilG, PilH, and ChpArec. Reactions were initiated by addition of 30 μ m [γ- 32 P]ATP to mixtures

Techniques Used:

Reactivities of the ChpA Xpts with ATP in the presence of the HATPase_c catalytic domain. ChpA AA− (1.9 μ m ) was incubated with each Xpt (Hpt1–Hpt6, Spt, and Tpt present at 40 μ m ) and [γ- 32 P]ATP (150 μ m )
Figure Legend Snippet: Reactivities of the ChpA Xpts with ATP in the presence of the HATPase_c catalytic domain. ChpA AA− (1.9 μ m ) was incubated with each Xpt (Hpt1–Hpt6, Spt, and Tpt present at 40 μ m ) and [γ- 32 P]ATP (150 μ m )

Techniques Used: Incubation, Single-particle Tracking

53) Product Images from "Phosphorylation of TGB1 by protein kinase CK2 promotes barley stripe mosaic virus movement in monocots and dicots"

Article Title: Phosphorylation of TGB1 by protein kinase CK2 promotes barley stripe mosaic virus movement in monocots and dicots

Journal: Journal of Experimental Botany

doi: 10.1093/jxb/erv237

Phosphorylation of the XJ TGB1 protein in vitro and in vivo . (A) Coomasie Brilliant Blue (CBB) staining of recombinant XJ TGB1 protein purified from E. coli cells. Molecular weight markers (Fermentas) are indicated on the left side of the gel. (B) In vitro phosphorylation of purified XJ TGB1 protein by cellular kinases present in healthy N. benthamiana extracts in the absence or presence of [γ- 32 P]ATP or [γ- 32 P]GTP. After the phosphorylation reactions, the TGB1 proteins were separated by 12.5% SDS-PAGE and the incorporated radioactivity was analysed by autoradiography. Reaction mixtures lacking XJ TGB1 protein or N. benthamiana protein extracts served as negative controls. The CBB staining in the lower panel indicates that similar amounts of the XJ TGB1 protein were present in each in vitro phosphorylation reaction. (C) In vivo phosphorylation of XJ TGB1 protein in N. benthamiana by Western blotting with α-TGB1 polyclonal antibodies and α-threonine antibodies. A mock agroinfiltration lacking XJ RNAβ was used as a negative control and molecular weight markers (Thermo Scientific) were used to estimate the size of the XJ TGB1 protein. (D) In vivo phosphorylation of XJ TGB1 protein immunoprecipitated (IP) from N. benthamiana was analysed as in Fig. 2C . (This figure is available in colour at JXB online.)
Figure Legend Snippet: Phosphorylation of the XJ TGB1 protein in vitro and in vivo . (A) Coomasie Brilliant Blue (CBB) staining of recombinant XJ TGB1 protein purified from E. coli cells. Molecular weight markers (Fermentas) are indicated on the left side of the gel. (B) In vitro phosphorylation of purified XJ TGB1 protein by cellular kinases present in healthy N. benthamiana extracts in the absence or presence of [γ- 32 P]ATP or [γ- 32 P]GTP. After the phosphorylation reactions, the TGB1 proteins were separated by 12.5% SDS-PAGE and the incorporated radioactivity was analysed by autoradiography. Reaction mixtures lacking XJ TGB1 protein or N. benthamiana protein extracts served as negative controls. The CBB staining in the lower panel indicates that similar amounts of the XJ TGB1 protein were present in each in vitro phosphorylation reaction. (C) In vivo phosphorylation of XJ TGB1 protein in N. benthamiana by Western blotting with α-TGB1 polyclonal antibodies and α-threonine antibodies. A mock agroinfiltration lacking XJ RNAβ was used as a negative control and molecular weight markers (Thermo Scientific) were used to estimate the size of the XJ TGB1 protein. (D) In vivo phosphorylation of XJ TGB1 protein immunoprecipitated (IP) from N. benthamiana was analysed as in Fig. 2C . (This figure is available in colour at JXB online.)

Techniques Used: In Vitro, In Vivo, Staining, Recombinant, Purification, Molecular Weight, SDS Page, Radioactivity, Autoradiography, Western Blot, Negative Control, Immunoprecipitation

Thr-401 is the major XJ TGB1 protein site for CK2 phosphorylation. (A) LC-MS/MS analysis of XJ TGB1 protein phosphorylation by NbCK2α. The absence of phosphoric acid (97.9769Da) on the y 16 ion fragment demonstrates that Thr-401 is a phosphorylation site for CK2 kinase. (B) Identification of the phosphorylation sites in XJ TGB1 protein mutants by in vitro phosphorylation with HvCK2α and NbCK2α. The radioactive intensities of the XJ TGB1 protein and its phosphorylation mutants indicate the extent of radiolabelling with [γ- 32 P]ATP. CBB-stained proteins at the bottom of the panels (B) and (C) are as indicated in Fig. 2B . (C) Phosphorylation comparisons of selected XJ TGB1 protein mutants with wt XJ TGB1 protein to confirm that Thr-401 is the major phosphorylated residue. (This figure is available in colour at JXB online.)
Figure Legend Snippet: Thr-401 is the major XJ TGB1 protein site for CK2 phosphorylation. (A) LC-MS/MS analysis of XJ TGB1 protein phosphorylation by NbCK2α. The absence of phosphoric acid (97.9769Da) on the y 16 ion fragment demonstrates that Thr-401 is a phosphorylation site for CK2 kinase. (B) Identification of the phosphorylation sites in XJ TGB1 protein mutants by in vitro phosphorylation with HvCK2α and NbCK2α. The radioactive intensities of the XJ TGB1 protein and its phosphorylation mutants indicate the extent of radiolabelling with [γ- 32 P]ATP. CBB-stained proteins at the bottom of the panels (B) and (C) are as indicated in Fig. 2B . (C) Phosphorylation comparisons of selected XJ TGB1 protein mutants with wt XJ TGB1 protein to confirm that Thr-401 is the major phosphorylated residue. (This figure is available in colour at JXB online.)

Techniques Used: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, In Vitro, Staining

54) Product Images from "High CO2 Leads to Na,K-ATPase Endocytosis via c-Jun Amino-Terminal Kinase-Induced LMO7b Phosphorylation"

Article Title: High CO2 Leads to Na,K-ATPase Endocytosis via c-Jun Amino-Terminal Kinase-Induced LMO7b Phosphorylation

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00813-15

LMO7b is a target for JNK phosphorylation. (A) In vitro JNK kinase assay was performed by incubating purified recombinant JNK1 (150 ng) with LMO7b immunoprecipitated (IP) from rATII cells in the presence or absence of [γ- 32 P]ATP. A representative
Figure Legend Snippet: LMO7b is a target for JNK phosphorylation. (A) In vitro JNK kinase assay was performed by incubating purified recombinant JNK1 (150 ng) with LMO7b immunoprecipitated (IP) from rATII cells in the presence or absence of [γ- 32 P]ATP. A representative

Techniques Used: In Vitro, Kinase Assay, Purification, Recombinant, Immunoprecipitation

55) Product Images from "Proteolysis of histidine kinase VgrS inhibits its autophosphorylation and promotes osmostress resistance in Xanthomonas campestris"

Article Title: Proteolysis of histidine kinase VgrS inhibits its autophosphorylation and promotes osmostress resistance in Xanthomonas campestris

Journal: Nature Communications

doi: 10.1038/s41467-018-07228-4

Prc regulates the VgrR–promoter-binding interactions in bacterial cells. a Bacterial growth on NYG–sorbitol plate. Bacterial cultures were serially diluted and inoculated onto NYG plus 1.0 M sorbitol plate and grown for 72 h at 28 °C. The experiment was repeated three times. b and c Electrophoretic mobility shift assays revealed that VgrR directly bound the promoter regions of XC_0943 and yciE . PCR products of the promoter regions were labelled with [γ- 32 P]ATP and used as DNA probes. Unlabelled DNA and non-specific DNA were used as competitors. The sequence of the DNA probe is shown below with the VgrR-binding motif in magenta. Numbers indicate the location relative to the translation initiation site. Each experiment was repeated two times. Triangles indicate VgrR–DNA complexes. d and e The prc mutation caused decreases in the transcription levels of XC_0943 and yciE when bacterial strains were grown under osmostress. qRT-PCR was used to quantify the mRNA levels of these genes in different bacterial strains before and after osmostress stimulation (1.0 M sorbitol, 5 min). Amplification of the cDNA of tmRNA was used as an internal control. A representative of three independent experiments is shown. f and g The prc mutation caused decreases in VgrR–DNA binding in bacterial cells. ChIP-qPCR was conducted to quantify the enrichment of VgrR at the promoter regions of XC_0943 and yciE in vivo when bacterial strains were grown under osmostress conditions (NYG medium plus 1.0 M sorbitol for 5 min). The experiment was repeated three times. In d – g Error bars indicate the standard deviations. Asterisks indicate significant differences of strains before and after osmostress (Student’s t -test, P
Figure Legend Snippet: Prc regulates the VgrR–promoter-binding interactions in bacterial cells. a Bacterial growth on NYG–sorbitol plate. Bacterial cultures were serially diluted and inoculated onto NYG plus 1.0 M sorbitol plate and grown for 72 h at 28 °C. The experiment was repeated three times. b and c Electrophoretic mobility shift assays revealed that VgrR directly bound the promoter regions of XC_0943 and yciE . PCR products of the promoter regions were labelled with [γ- 32 P]ATP and used as DNA probes. Unlabelled DNA and non-specific DNA were used as competitors. The sequence of the DNA probe is shown below with the VgrR-binding motif in magenta. Numbers indicate the location relative to the translation initiation site. Each experiment was repeated two times. Triangles indicate VgrR–DNA complexes. d and e The prc mutation caused decreases in the transcription levels of XC_0943 and yciE when bacterial strains were grown under osmostress. qRT-PCR was used to quantify the mRNA levels of these genes in different bacterial strains before and after osmostress stimulation (1.0 M sorbitol, 5 min). Amplification of the cDNA of tmRNA was used as an internal control. A representative of three independent experiments is shown. f and g The prc mutation caused decreases in VgrR–DNA binding in bacterial cells. ChIP-qPCR was conducted to quantify the enrichment of VgrR at the promoter regions of XC_0943 and yciE in vivo when bacterial strains were grown under osmostress conditions (NYG medium plus 1.0 M sorbitol for 5 min). The experiment was repeated three times. In d – g Error bars indicate the standard deviations. Asterisks indicate significant differences of strains before and after osmostress (Student’s t -test, P

Techniques Used: Binding Assay, Electrophoretic Mobility Shift Assay, Polymerase Chain Reaction, Sequencing, Mutagenesis, Quantitative RT-PCR, Amplification, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, In Vivo

prc controls the VgrR regulon and VgrR promoter-binding landscapes. a Venn diagram showing the number of genes with promoters that potentially bound to VgrR. ChIP-seq was used to identify the VgrR-binding DNAs. Genes identified from the WT strain and the prc mutant are shown. b Predicted consensus VgrR-binding DNA motif based on ChIP-seq data. Weblogo was used to show the nucleotide composition. c Functional classification of the VgrR-regulated genes identified by ChIP-seq. Gene details are listed in Supplementary Table 4 . d An electrophoretic mobility shift assay revealed that VgrR directly bound the promoter region of prc . PCR products of the promoter region were labelled with [γ- 32 P]ATP and used as DNA probes. Unlabelled DNA and unspecific DNA were used as competitors. The sequence of the DNA probe is shown below, with the VgrR-binding motif in magenta. Numbers indicate the location relative to the translation initiation site. Each experiment was repeated two times. Triangle indicates VgrR–DNA complexes. e vgrR positively controls the transcription of prc . qRT-PCR was used to quantify prc mRNA in different bacterial strains before and after osmostress stimulation (1.0 M sorbitol, 5 min). Amplification of the cDNA of tmRNA was used as an internal control. A representative of three independent experiments is shown. f Deletion of prc decreases VgrR-promoter binding in bacterial cells. ChIP-qPCR was employed to quantify the enrichment of VgrR at the promoter region of prc under osmostress growth conditions (NYG medium plus 1.0 M sorbitol for 5 min) or no stimulation. The experiment was repeated three times. In e and f , error bars indicate the standard deviations. Asterisks indicate significant differences relative to the WT strain (Student’s t -test, P
Figure Legend Snippet: prc controls the VgrR regulon and VgrR promoter-binding landscapes. a Venn diagram showing the number of genes with promoters that potentially bound to VgrR. ChIP-seq was used to identify the VgrR-binding DNAs. Genes identified from the WT strain and the prc mutant are shown. b Predicted consensus VgrR-binding DNA motif based on ChIP-seq data. Weblogo was used to show the nucleotide composition. c Functional classification of the VgrR-regulated genes identified by ChIP-seq. Gene details are listed in Supplementary Table 4 . d An electrophoretic mobility shift assay revealed that VgrR directly bound the promoter region of prc . PCR products of the promoter region were labelled with [γ- 32 P]ATP and used as DNA probes. Unlabelled DNA and unspecific DNA were used as competitors. The sequence of the DNA probe is shown below, with the VgrR-binding motif in magenta. Numbers indicate the location relative to the translation initiation site. Each experiment was repeated two times. Triangle indicates VgrR–DNA complexes. e vgrR positively controls the transcription of prc . qRT-PCR was used to quantify prc mRNA in different bacterial strains before and after osmostress stimulation (1.0 M sorbitol, 5 min). Amplification of the cDNA of tmRNA was used as an internal control. A representative of three independent experiments is shown. f Deletion of prc decreases VgrR-promoter binding in bacterial cells. ChIP-qPCR was employed to quantify the enrichment of VgrR at the promoter region of prc under osmostress growth conditions (NYG medium plus 1.0 M sorbitol for 5 min) or no stimulation. The experiment was repeated three times. In e and f , error bars indicate the standard deviations. Asterisks indicate significant differences relative to the WT strain (Student’s t -test, P

Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Mutagenesis, Functional Assay, Electrophoretic Mobility Shift Assay, Polymerase Chain Reaction, Sequencing, Quantitative RT-PCR, Amplification, Real-time Polymerase Chain Reaction

Prc cleaves the sensor region of VgrS to inhibit its autokinase activity. a – c Prc inhibited full-length VgrS autophosphorylation in a protease activity-dependent manner. a Full-length VgrS embedded in the inverted membrane vesicles (IMV, 10 μM) was phosphorylated by [γ- 32 P]ATP. Before the addition of 10 μCi [γ- 32 P]ATP, active Prc or inactive PrcS 475A (2 μM) was added into the mixture. VgrS H186A IMV was used as a negative control of phosphorylation. b Prc did not affect the autophosphorylation of a soluble, cytosolic fragment of VgrS. In total, 10 μM soluble VgrS containing the transmitter region (MBP-VgrS cyto ) was used in the assay. c Prc inhibited the phosphorylation level of VgrR. After VgrS autophosphorylation, 10 μM VgrR was added into the mixture to elicit the VgrS–VgrR phosphotransfer reaction. In a , b and c : Upper panels show autophosphorylation assays; lower panels show Coomassie bright blue-stained gels used to check the amount of loaded protein. Aliquots were removed from the mixture at the indicated time points. The reaction was stopped by 6X SDS buffer, separated by SDS-PAGE and analysed by autoradiography. d and e Prc degraded the VgrS sensor in vitro. The purified VgrS sensor (100 μM) was co-incubated with 5 μM active Prc d or inactive Prc S475A e in enzymatic reaction buffer at 28 °C for the indicated time. Reactions were stopped, and the products were analysed by SDS-PAGE together with silver staining. f Western blotting revealed that Prc degraded N-terminal HA-tags in vivo. A bacterial strain that encoded recombinant VgrS fused with an HA-tag between the 6th and 7th residues was constructed independently in the ΔvgrS background. The bacterial strain was stimulated by 1.0 M sorbitol for different time periods. Total proteins were extracted, separated by SDS-PAGE, and analysed by western blotting. A polyclonal antibody of VgrS (α-VgrS) was used to measure the amount of VgrS protein, while monoclonal HA antibody (α-HA) was used to detect the N-terminal region of VgrS that was potentially cleaved by Prc, and the polyclonal antibody of RNAP (α-RNAP) was used as internal control. a – f , the experiments were repeated three times
Figure Legend Snippet: Prc cleaves the sensor region of VgrS to inhibit its autokinase activity. a – c Prc inhibited full-length VgrS autophosphorylation in a protease activity-dependent manner. a Full-length VgrS embedded in the inverted membrane vesicles (IMV, 10 μM) was phosphorylated by [γ- 32 P]ATP. Before the addition of 10 μCi [γ- 32 P]ATP, active Prc or inactive PrcS 475A (2 μM) was added into the mixture. VgrS H186A IMV was used as a negative control of phosphorylation. b Prc did not affect the autophosphorylation of a soluble, cytosolic fragment of VgrS. In total, 10 μM soluble VgrS containing the transmitter region (MBP-VgrS cyto ) was used in the assay. c Prc inhibited the phosphorylation level of VgrR. After VgrS autophosphorylation, 10 μM VgrR was added into the mixture to elicit the VgrS–VgrR phosphotransfer reaction. In a , b and c : Upper panels show autophosphorylation assays; lower panels show Coomassie bright blue-stained gels used to check the amount of loaded protein. Aliquots were removed from the mixture at the indicated time points. The reaction was stopped by 6X SDS buffer, separated by SDS-PAGE and analysed by autoradiography. d and e Prc degraded the VgrS sensor in vitro. The purified VgrS sensor (100 μM) was co-incubated with 5 μM active Prc d or inactive Prc S475A e in enzymatic reaction buffer at 28 °C for the indicated time. Reactions were stopped, and the products were analysed by SDS-PAGE together with silver staining. f Western blotting revealed that Prc degraded N-terminal HA-tags in vivo. A bacterial strain that encoded recombinant VgrS fused with an HA-tag between the 6th and 7th residues was constructed independently in the ΔvgrS background. The bacterial strain was stimulated by 1.0 M sorbitol for different time periods. Total proteins were extracted, separated by SDS-PAGE, and analysed by western blotting. A polyclonal antibody of VgrS (α-VgrS) was used to measure the amount of VgrS protein, while monoclonal HA antibody (α-HA) was used to detect the N-terminal region of VgrS that was potentially cleaved by Prc, and the polyclonal antibody of RNAP (α-RNAP) was used as internal control. a – f , the experiments were repeated three times

Techniques Used: Activity Assay, Negative Control, Staining, SDS Page, Autoradiography, In Vitro, Purification, Incubation, Silver Staining, Western Blot, In Vivo, Recombinant, Construct

56) Product Images from "Adenine DNA Glycosylase Activity of 14 Human MutY Homolog (MUTYH) Variant Proteins Found in Patients with Colorectal Polyposis and Cancer"

Article Title: Adenine DNA Glycosylase Activity of 14 Human MutY Homolog (MUTYH) Variant Proteins Found in Patients with Colorectal Polyposis and Cancer

Journal: Human Mutation

doi: 10.1002/humu.21363

Measurement of DNA glycosylase activity of MUTYH type 1 protein. ( A ) Purification of wild-type MUTYH type 1 protein resolved by SDS-polyacrylamide gel electrophoresis (PAGE) and stained with Coomassie Brilliant Blue (CBB). The human MUTYH type 1 cDNA sequence was inserted into a pET25b(+) expression vector (Novagen, Darmstadt, Germany). E. coli BL21-CodonPlus (DE3)-RP competent cells (Stratagene, La Jolla, CA) were transformed with the MUTYH-pET25b vector and cultured at 37°C until an A 600 of 0.6. After incubation with 0.1 mM IPTG at 20°C for 12 h, MUTYH-His 6 protein was purified with TALON metal affinity resins (Clontech, Palo Alto, CA) and a TALON 2-ml disposable gravity column (Clontech). The protein was then dialyzed against buffer containing 10 mM sodium phosphate (pH 7.6), 50 mM NaCl, 0.5 mM DTT, 0.1 mM EDTA, 0.5 mM PMSF, 2 μg/ml pepstatin, 2 μg/ml leupeptin, 50 μM chymostatin, and 10% glycerol. Lysates of E. coli culture without or with IPTG induction and purified MUTYH type 1 protein are shown. The arrow points to the MUTYH-His 6 protein band. ( B ) Western blot of purified wild-type MUTYH type 1 protein tagged with His 6 . Purified recombinant NEIL1-His 6 protein, which was prepared by using pET25b(+) vector (Novagen) and E. coli BL21-CodonPlus (DE3)-RP cells (Stratagene) previously ( Shinmura et al., 2004 ), was included as a negative control. Purified recombinant protein was mixed with an equal volume of 2x SDS sample buffer and boiled. A 2 ug protein was subjected to SDS-PAGE and electrophoretically transferred to a polyvinylidene difluoride membrane (GE Healthcare Bio-Science Corp., Piscataway,NJ). The membrane was blocked with nonfat milk and incubated with an anti-MUTYH polyclonal antibody ( Ohtsubo et al., 2000 ). After washing, the membrane was incubated with an anti-rabbit HRP-conjugated secondary antibody (GE Healthcare Bio-Science Corp.). The membrane was then washed, and immunoreactivity was visualized with an ECL Plus chemiluminescence system (GE Healthcare Bio-Science Corp.). MUTYH-His 6 protein is indicated by the arrow. ( C ) The DNA glycosylase activity of wild-type MUTYH type 1 protein on double-stranded DNA containing an A:8-hydroxyguanine (8-OHG). 30-mer oligonucleotides containing and not containing a single 8-OHG (5′-CTG GTG GCC TGA C[8-OHG or T]C ATT CCC CAA CTA GTG-3′) were chemically synthesized and purified by PAGE (Japan Bio Services, Saitama, Japan). Complementary oligonucleotides containing an adenine opposite the 8-OHG or T were 32 P-labeled at the 5′ terminus with a MEGALABEL kit (Takara, Osaka, Japan) and a [γ- 32 P]ATP (PerkinElmer, Tokyo, Japan), and then annealed to oligonucleotides containing a single 8-OHG or T. The reaction mixture containing 20 mM sodium phosphate (pH 7.6), 100 mM NaCl, 0.5 mM DTT, 0.5 mM EDTA, 5 μM ZnCl 2 , 1.5% glycerol, 2.5 nM labeled oligonucleotide, 50 μg/ml BSA, and purified MUTYH protein was incubated at 37°C, and the mixture was treated with 0.1 M NaOH at 95°C for 4 min. After adding denaturing formamide dye to the mixture, it was heated at 95°C for 3 min, and subjected to 20% PAGE. A 32 P-labeled marker oligonucleotide was used as a size marker for the cleavage products. The radioactivity of intact and cleaved oligonucleotides was quantified by using an FLA-3000 fluoroimage analyzer (Fuji Film, Tokyo, Japan) and ImageGauge software (Fuji Film) ( Goto et al., 2009 ). The intact 30-mer oligonucleotides and cleavage products are indicated by the arrows. Heat-inactivation of the MUTYH protein was accomplished by heating the protein at 100°C for 5 min. 8G means 8-hydroxyguanine. ( D ) Time-course assay of cleavage of DNA containing an A:8-OHG by wild-type MUTYH type 1 protein. The MUTYH type 1 protein (260 fmole) was incubated at 37°C for 0 - 60 min with double-stranded oligonucleotide containing an A:8-OHG (50 fmole). The amount of cleavage products as a proportion of total oligonucleotides was calculated as % incision. The % incision values are shown as means ± standard errors of data from three independent experiments. ( E ) DNA glycosylase activities of wild-type MUTYH type 1 protein and their variant proteins on an A:8-OHG substrate. DNA cleavage activities of MUTYH type 1 proteins were measured at 37°C for 15 min. The amount of cleavage products as a proportion of total oligonucleotides was calculated as % incision, and the % incision of each variant-type MUTYH protein is shown relative to that of wild-type (WT) MUTYH protein, which has been set equal to 100. Values are means ± standard errors of data from three independent experiments. ND, not detected.
Figure Legend Snippet: Measurement of DNA glycosylase activity of MUTYH type 1 protein. ( A ) Purification of wild-type MUTYH type 1 protein resolved by SDS-polyacrylamide gel electrophoresis (PAGE) and stained with Coomassie Brilliant Blue (CBB). The human MUTYH type 1 cDNA sequence was inserted into a pET25b(+) expression vector (Novagen, Darmstadt, Germany). E. coli BL21-CodonPlus (DE3)-RP competent cells (Stratagene, La Jolla, CA) were transformed with the MUTYH-pET25b vector and cultured at 37°C until an A 600 of 0.6. After incubation with 0.1 mM IPTG at 20°C for 12 h, MUTYH-His 6 protein was purified with TALON metal affinity resins (Clontech, Palo Alto, CA) and a TALON 2-ml disposable gravity column (Clontech). The protein was then dialyzed against buffer containing 10 mM sodium phosphate (pH 7.6), 50 mM NaCl, 0.5 mM DTT, 0.1 mM EDTA, 0.5 mM PMSF, 2 μg/ml pepstatin, 2 μg/ml leupeptin, 50 μM chymostatin, and 10% glycerol. Lysates of E. coli culture without or with IPTG induction and purified MUTYH type 1 protein are shown. The arrow points to the MUTYH-His 6 protein band. ( B ) Western blot of purified wild-type MUTYH type 1 protein tagged with His 6 . Purified recombinant NEIL1-His 6 protein, which was prepared by using pET25b(+) vector (Novagen) and E. coli BL21-CodonPlus (DE3)-RP cells (Stratagene) previously ( Shinmura et al., 2004 ), was included as a negative control. Purified recombinant protein was mixed with an equal volume of 2x SDS sample buffer and boiled. A 2 ug protein was subjected to SDS-PAGE and electrophoretically transferred to a polyvinylidene difluoride membrane (GE Healthcare Bio-Science Corp., Piscataway,NJ). The membrane was blocked with nonfat milk and incubated with an anti-MUTYH polyclonal antibody ( Ohtsubo et al., 2000 ). After washing, the membrane was incubated with an anti-rabbit HRP-conjugated secondary antibody (GE Healthcare Bio-Science Corp.). The membrane was then washed, and immunoreactivity was visualized with an ECL Plus chemiluminescence system (GE Healthcare Bio-Science Corp.). MUTYH-His 6 protein is indicated by the arrow. ( C ) The DNA glycosylase activity of wild-type MUTYH type 1 protein on double-stranded DNA containing an A:8-hydroxyguanine (8-OHG). 30-mer oligonucleotides containing and not containing a single 8-OHG (5′-CTG GTG GCC TGA C[8-OHG or T]C ATT CCC CAA CTA GTG-3′) were chemically synthesized and purified by PAGE (Japan Bio Services, Saitama, Japan). Complementary oligonucleotides containing an adenine opposite the 8-OHG or T were 32 P-labeled at the 5′ terminus with a MEGALABEL kit (Takara, Osaka, Japan) and a [γ- 32 P]ATP (PerkinElmer, Tokyo, Japan), and then annealed to oligonucleotides containing a single 8-OHG or T. The reaction mixture containing 20 mM sodium phosphate (pH 7.6), 100 mM NaCl, 0.5 mM DTT, 0.5 mM EDTA, 5 μM ZnCl 2 , 1.5% glycerol, 2.5 nM labeled oligonucleotide, 50 μg/ml BSA, and purified MUTYH protein was incubated at 37°C, and the mixture was treated with 0.1 M NaOH at 95°C for 4 min. After adding denaturing formamide dye to the mixture, it was heated at 95°C for 3 min, and subjected to 20% PAGE. A 32 P-labeled marker oligonucleotide was used as a size marker for the cleavage products. The radioactivity of intact and cleaved oligonucleotides was quantified by using an FLA-3000 fluoroimage analyzer (Fuji Film, Tokyo, Japan) and ImageGauge software (Fuji Film) ( Goto et al., 2009 ). The intact 30-mer oligonucleotides and cleavage products are indicated by the arrows. Heat-inactivation of the MUTYH protein was accomplished by heating the protein at 100°C for 5 min. 8G means 8-hydroxyguanine. ( D ) Time-course assay of cleavage of DNA containing an A:8-OHG by wild-type MUTYH type 1 protein. The MUTYH type 1 protein (260 fmole) was incubated at 37°C for 0 - 60 min with double-stranded oligonucleotide containing an A:8-OHG (50 fmole). The amount of cleavage products as a proportion of total oligonucleotides was calculated as % incision. The % incision values are shown as means ± standard errors of data from three independent experiments. ( E ) DNA glycosylase activities of wild-type MUTYH type 1 protein and their variant proteins on an A:8-OHG substrate. DNA cleavage activities of MUTYH type 1 proteins were measured at 37°C for 15 min. The amount of cleavage products as a proportion of total oligonucleotides was calculated as % incision, and the % incision of each variant-type MUTYH protein is shown relative to that of wild-type (WT) MUTYH protein, which has been set equal to 100. Values are means ± standard errors of data from three independent experiments. ND, not detected.

Techniques Used: Activity Assay, Purification, Polyacrylamide Gel Electrophoresis, Staining, Sequencing, Expressing, Plasmid Preparation, Transformation Assay, Cell Culture, Incubation, Western Blot, Recombinant, Negative Control, SDS Page, CTG Assay, Countercurrent Chromatography, Cellular Antioxidant Activity Assay, Synthesized, Labeling, Marker, Radioactivity, Software, Variant Assay

57) Product Images from "Dynamic Ubiquitination of the Mitogen-activated Protein Kinase Kinase (MAPKK) Ste7 Determines Mitogen-activated Protein Kinase (MAPK) Specificity *Dynamic Ubiquitination of the Mitogen-activated Protein Kinase Kinase (MAPKK) Ste7 Determines Mitogen-activated Protein Kinase (MAPK) Specificity * ♦"

Article Title: Dynamic Ubiquitination of the Mitogen-activated Protein Kinase Kinase (MAPKK) Ste7 Determines Mitogen-activated Protein Kinase (MAPK) Specificity *Dynamic Ubiquitination of the Mitogen-activated Protein Kinase Kinase (MAPKK) Ste7 Determines Mitogen-activated Protein Kinase (MAPK) Specificity * ♦

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M113.475707

Fus3 phosphorylates Ubp3 to limit pheromone-stimulated MAP kinase activation. A, in vitro kinase assays were performed using Fus3 purified from E. coli , FLAG-tagged Ubp3, or the phospho-site mutant Ubp3 S695A purified from yeast and [γ- 32 P]ATP
Figure Legend Snippet: Fus3 phosphorylates Ubp3 to limit pheromone-stimulated MAP kinase activation. A, in vitro kinase assays were performed using Fus3 purified from E. coli , FLAG-tagged Ubp3, or the phospho-site mutant Ubp3 S695A purified from yeast and [γ- 32 P]ATP

Techniques Used: Activation Assay, In Vitro, Purification, Mutagenesis

58) Product Images from "Casein Kinase 2 Reverses Tail-Independent Inactivation of Kinesin-1"

Article Title: Casein Kinase 2 Reverses Tail-Independent Inactivation of Kinesin-1

Journal: Nature communications

doi: 10.1038/ncomms1760

Role of CK2 kinase activity in K560 activation. ( a ) Autoradiogram of CK2/native kinesin mixture, supplemented with 1 μCi of [γ- 32 P]-ATP. Control experiments lacking CK2 are shown. ( b ) Effect of incubation temperature on CK2 kinase activity in vitro , assayed by autoradiogram. Parallel samples of wild type K560/CK2 mixture, supplemented with 1 μCi of [γ- 32 P]-ATP, were incubated on ice (1.5 hr) or at 30°C (40 min). Radioactive phosphate incorporation in both K560 and the CK2 itself were severely limited when incubated on ice. ( c ) Effect of the CK2 specific inhibitor, TBCA (100 μM), on the kinase’s ability to phosphorylate wild type K560, assayed by autoradiogram. Diminished CK2 auto-phosphorylation (in ‘CK2’ band) by TBCA-treatment further verified the pharmacological inhibition of kinase activity. ( d ) LC-MS/MS spectra of tryptic phosphopeptide (TKEYELLS(phospho)DELNQK) and non-phosphopeptide (TKEYELLSDELNQK). Ion count intensity in the phosphopeptide spectrum is enhanced in the indicated regions for comparison with the non-phosphopeptide. We obtain 81% sequence coverage of K560 protein using LC-MS/MS analysis. ( e ) Schematic (left) of wild-type K560and phospho-mutant S52A, and autoradiogram (right) assaying the ability of CK2 to phosphorylate the mutant (‘S520A’) vs. wild type motor (‘WT’). ( f ) Motor co-sedimentation with microtubules at 4 mM AMPPNP, assayed by immunoblot. Both the phosphor-mutant (‘S520A’) and the wild-type motor (‘WT K560’) were incubated with and without CK2 (3:1 CK2:motor) for 40 min at 30 °C prior to microtubule pulldowns. For ‘WT K560’, we introduced the CK2 specific kinase inhibitor, TBCA (100μM), during motor/kinase incubation.
Figure Legend Snippet: Role of CK2 kinase activity in K560 activation. ( a ) Autoradiogram of CK2/native kinesin mixture, supplemented with 1 μCi of [γ- 32 P]-ATP. Control experiments lacking CK2 are shown. ( b ) Effect of incubation temperature on CK2 kinase activity in vitro , assayed by autoradiogram. Parallel samples of wild type K560/CK2 mixture, supplemented with 1 μCi of [γ- 32 P]-ATP, were incubated on ice (1.5 hr) or at 30°C (40 min). Radioactive phosphate incorporation in both K560 and the CK2 itself were severely limited when incubated on ice. ( c ) Effect of the CK2 specific inhibitor, TBCA (100 μM), on the kinase’s ability to phosphorylate wild type K560, assayed by autoradiogram. Diminished CK2 auto-phosphorylation (in ‘CK2’ band) by TBCA-treatment further verified the pharmacological inhibition of kinase activity. ( d ) LC-MS/MS spectra of tryptic phosphopeptide (TKEYELLS(phospho)DELNQK) and non-phosphopeptide (TKEYELLSDELNQK). Ion count intensity in the phosphopeptide spectrum is enhanced in the indicated regions for comparison with the non-phosphopeptide. We obtain 81% sequence coverage of K560 protein using LC-MS/MS analysis. ( e ) Schematic (left) of wild-type K560and phospho-mutant S52A, and autoradiogram (right) assaying the ability of CK2 to phosphorylate the mutant (‘S520A’) vs. wild type motor (‘WT’). ( f ) Motor co-sedimentation with microtubules at 4 mM AMPPNP, assayed by immunoblot. Both the phosphor-mutant (‘S520A’) and the wild-type motor (‘WT K560’) were incubated with and without CK2 (3:1 CK2:motor) for 40 min at 30 °C prior to microtubule pulldowns. For ‘WT K560’, we introduced the CK2 specific kinase inhibitor, TBCA (100μM), during motor/kinase incubation.

Techniques Used: Activity Assay, Activation Assay, Incubation, In Vitro, Inhibition, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Sequencing, Mutagenesis, Sedimentation

59) Product Images from "Heat shock protein 20 (HSP20) is a novel substrate for protein kinase D1 (PKD1)) Heat shock protein 20 (HSP20) is a novel substrate for protein kinase D1 (PKD1)"

Article Title: Heat shock protein 20 (HSP20) is a novel substrate for protein kinase D1 (PKD1)) Heat shock protein 20 (HSP20) is a novel substrate for protein kinase D1 (PKD1)

Journal: Cell Biochemistry and Function

doi: 10.1002/cbf.3147

Identification of PKD1–HSP20 interaction and phosphorylation sites. (A) HSP20 is shown schematically with phosphorylation site (P), WDPF domain (dark shaded area), conserved region (light shaded area) and α‐crystallin domain (black area). Peptide array analysis identifies PKD1 binding sites on HSP20. (B) The key residues involved in the HSP20–PKD1 interaction are delineated using alanine scanning peptide arrays where each residue is sequentially substituted by alanine (or aspartate if residue is alanine). (C) In vitro phosphorylation assay on peptide array using [γ‐ 32 P‐ATP] revealed that HSP20 is phosphorylated at Ser16 by PKD1. (D) In vitro kinase assay (cold) using purified His‐HSP20 and PKD1 active protein further verified HSP20 phosphorylation by PKD1
Figure Legend Snippet: Identification of PKD1–HSP20 interaction and phosphorylation sites. (A) HSP20 is shown schematically with phosphorylation site (P), WDPF domain (dark shaded area), conserved region (light shaded area) and α‐crystallin domain (black area). Peptide array analysis identifies PKD1 binding sites on HSP20. (B) The key residues involved in the HSP20–PKD1 interaction are delineated using alanine scanning peptide arrays where each residue is sequentially substituted by alanine (or aspartate if residue is alanine). (C) In vitro phosphorylation assay on peptide array using [γ‐ 32 P‐ATP] revealed that HSP20 is phosphorylated at Ser16 by PKD1. (D) In vitro kinase assay (cold) using purified His‐HSP20 and PKD1 active protein further verified HSP20 phosphorylation by PKD1

Techniques Used: Peptide Microarray, Binding Assay, In Vitro, Phosphorylation Assay, Kinase Assay, Purification

60) Product Images from "Functional Analysis of the VirSR Phosphorelay from Clostridium perfringens"

Article Title: Functional Analysis of the VirSR Phosphorelay from Clostridium perfringens

Journal: PLoS ONE

doi: 10.1371/journal.pone.0005849

Autophosphorylation of VirSc and its derivatives. (A) In the absence of purified VirSc, a labelled protein band was not observed in the presence of [γ- 32 P]ATP (lane 1) or [α- 32 P]ATP (lane 2). A labelled band of ∼27 kDa was observed when purified VirSc (20 µM) was incubated in phosphorylation buffer in the presence of [γ- 32 P]ATP (lane 3). To show specificity of autophosphorylation, VirSc was incubated with [α- 32 P]ATP (lane 4), or with [γ- 32 P]ATP in the presence of 50 mM EDTA (lane 5) or 2.5 mM unlabelled ATP (lane 6). (B) Autophosphorylation of substituted derivatives of VirSc. VirSc was able to autophosphorylate (lane 1), while VirSc H255I (lane 2), VirSc C335Y (lane 3) and VirSc G402D (lane 5) could not undergo autophosphorylation. VirSc G400A (lane 4) showed severely reduced autophosphorylation. The band corresponding to phosphorylated VirSc (∼27 kDa) is indicated by the arrow. Molecular size markers (kDa) are as indicated.
Figure Legend Snippet: Autophosphorylation of VirSc and its derivatives. (A) In the absence of purified VirSc, a labelled protein band was not observed in the presence of [γ- 32 P]ATP (lane 1) or [α- 32 P]ATP (lane 2). A labelled band of ∼27 kDa was observed when purified VirSc (20 µM) was incubated in phosphorylation buffer in the presence of [γ- 32 P]ATP (lane 3). To show specificity of autophosphorylation, VirSc was incubated with [α- 32 P]ATP (lane 4), or with [γ- 32 P]ATP in the presence of 50 mM EDTA (lane 5) or 2.5 mM unlabelled ATP (lane 6). (B) Autophosphorylation of substituted derivatives of VirSc. VirSc was able to autophosphorylate (lane 1), while VirSc H255I (lane 2), VirSc C335Y (lane 3) and VirSc G402D (lane 5) could not undergo autophosphorylation. VirSc G400A (lane 4) showed severely reduced autophosphorylation. The band corresponding to phosphorylated VirSc (∼27 kDa) is indicated by the arrow. Molecular size markers (kDa) are as indicated.

Techniques Used: Purification, Incubation

Phosphotransfer from VirSc to VirR. VirSc (10 µM) was incubated in phosphotransfer buffer in the presence of [γ- 32 P]ATP at room temperature before purified (A) VirR or (B) VirR D57N was added. Reactions were incubated at room temperature for the time indicated above each well. The first lane of each gel contains autophosphorylated VirSc, while subsequent lanes show the reactions with both phosphorylated VirSc and VirR or VirR D57N . The band corresponding to phosphorylated VirSc (∼27 kDa) or VirR (∼32 kDa) is indicated by the arrow or an asterisk, respectively. Molecular size markers (kDa) are as indicated.
Figure Legend Snippet: Phosphotransfer from VirSc to VirR. VirSc (10 µM) was incubated in phosphotransfer buffer in the presence of [γ- 32 P]ATP at room temperature before purified (A) VirR or (B) VirR D57N was added. Reactions were incubated at room temperature for the time indicated above each well. The first lane of each gel contains autophosphorylated VirSc, while subsequent lanes show the reactions with both phosphorylated VirSc and VirR or VirR D57N . The band corresponding to phosphorylated VirSc (∼27 kDa) or VirR (∼32 kDa) is indicated by the arrow or an asterisk, respectively. Molecular size markers (kDa) are as indicated.

Techniques Used: Incubation, Purification

61) Product Images from "The Vibrio cholerae Hybrid Sensor Kinase VieS Contributes to Motility and Biofilm Regulation by Altering the Cyclic Diguanylate Level ▿"

Article Title: The Vibrio cholerae Hybrid Sensor Kinase VieS Contributes to Motility and Biofilm Regulation by Altering the Cyclic Diguanylate Level ▿

Journal:

doi: 10.1128/JB.00541-08

VieS phosphorylates VieA at the D52 residue. (A) One micromolar MBP-′VieS was prelabeled by incubation for 30 min at 30°C in phosphorylation buffer in the presence of 30 μM [γ- 32 P]ATP. VieA was added to a 1 μM concentration,
Figure Legend Snippet: VieS phosphorylates VieA at the D52 residue. (A) One micromolar MBP-′VieS was prelabeled by incubation for 30 min at 30°C in phosphorylation buffer in the presence of 30 μM [γ- 32 P]ATP. VieA was added to a 1 μM concentration,

Techniques Used: Incubation, Concentration Assay

62) Product Images from "Transforming Growth Factor-?1 (TGF-?1) Regulates Cell Junction Restructuring via Smad-Mediated Repression and Clathrin-Mediated Endocytosis of Nectin-like Molecule 2 (Necl-2)"

Article Title: Transforming Growth Factor-?1 (TGF-?1) Regulates Cell Junction Restructuring via Smad-Mediated Repression and Clathrin-Mediated Endocytosis of Nectin-like Molecule 2 (Necl-2)

Journal: PLoS ONE

doi: 10.1371/journal.pone.0064316

EMSA of MyoD and CCAATa motifs and ChIP assay. (A) and (D) Dose-dependent and competition assay for EMSA of MyoD motif (A) and CCAATa motif (D). Double stranded oligonucleotides containing the respective motif were radiolabeled with [γ- 32 P]ATP and incubated with nuclear extract (1–15 µg) alone or in the presence of cold competitors (100- to 500-fold excess). (B–C) and (E–F) Labeled probes were incubated with vehicle or TGF-β1 treated nuclear extract (15 µg for MyoD motif and 10 µg for CCAATa motif) in the presence of specified antibodies or rabbit serum (Rb serum). (G) A schematic drawing illustrating the relative location of MyoD and CCAAT cis -acting motifs in the Necl-2 promoter and chromatin immunoprecipitation assay. TGF-β1-treated genomic DNA-protein samples were immunopreciptated with antibodies against Smad3 and Smad4 (2 µg) or rabbit serum. Precipitated DNA-protein complexes were subjected to DNA purification. The promoter region and the open reading frame of Necl-2 gene were amplified using specific primer pairs no. A287/A288 (for promoter region) and no. A107/A108 (for open reading frame), respectively, followed by agarose gel electrophoresis. Rb, Rabbit. Cpx, complex.
Figure Legend Snippet: EMSA of MyoD and CCAATa motifs and ChIP assay. (A) and (D) Dose-dependent and competition assay for EMSA of MyoD motif (A) and CCAATa motif (D). Double stranded oligonucleotides containing the respective motif were radiolabeled with [γ- 32 P]ATP and incubated with nuclear extract (1–15 µg) alone or in the presence of cold competitors (100- to 500-fold excess). (B–C) and (E–F) Labeled probes were incubated with vehicle or TGF-β1 treated nuclear extract (15 µg for MyoD motif and 10 µg for CCAATa motif) in the presence of specified antibodies or rabbit serum (Rb serum). (G) A schematic drawing illustrating the relative location of MyoD and CCAAT cis -acting motifs in the Necl-2 promoter and chromatin immunoprecipitation assay. TGF-β1-treated genomic DNA-protein samples were immunopreciptated with antibodies against Smad3 and Smad4 (2 µg) or rabbit serum. Precipitated DNA-protein complexes were subjected to DNA purification. The promoter region and the open reading frame of Necl-2 gene were amplified using specific primer pairs no. A287/A288 (for promoter region) and no. A107/A108 (for open reading frame), respectively, followed by agarose gel electrophoresis. Rb, Rabbit. Cpx, complex.

Techniques Used: Chromatin Immunoprecipitation, Competitive Binding Assay, Incubation, Labeling, DNA Purification, Amplification, Agarose Gel Electrophoresis

63) Product Images from "Inhibition of Mycoplasma pneumoniae growth by FDA-approved anticancer and antiviral nucleoside and nucleobase analogs"

Article Title: Inhibition of Mycoplasma pneumoniae growth by FDA-approved anticancer and antiviral nucleoside and nucleobase analogs

Journal: BMC Microbiology

doi: 10.1186/1471-2180-13-184

Substrate saturation curves of TFT with human TK2 (A), human TK1 (B), and Ureaplasma TK (C). Kinetic assays with TFT were performed by using [γ- 32 P]-ATP as the labelled substrate at fixed concentration and variable concentrations of TFT. The reaction products were separated by thin layer chromatography, and quantified as described in the experimental procedures. Data are from three independent measurements and are presented as mean ± SD.
Figure Legend Snippet: Substrate saturation curves of TFT with human TK2 (A), human TK1 (B), and Ureaplasma TK (C). Kinetic assays with TFT were performed by using [γ- 32 P]-ATP as the labelled substrate at fixed concentration and variable concentrations of TFT. The reaction products were separated by thin layer chromatography, and quantified as described in the experimental procedures. Data are from three independent measurements and are presented as mean ± SD.

Techniques Used: Concentration Assay, Thin Layer Chromatography

64) Product Images from "Staphylococcus aureus Methicillin-Resistance Factor fmtA Is Regulated by the Global Regulator SarA"

Article Title: Staphylococcus aureus Methicillin-Resistance Factor fmtA Is Regulated by the Global Regulator SarA

Journal: PLoS ONE

doi: 10.1371/journal.pone.0043998

DNaseI footprint analysis of SarA binding to seqA and seqB fragments of P fmtA . A. Each DNA sequence was labeled with [γ- 32 P]ATP on the 5′-end of the bottom strand.
Figure Legend Snippet: DNaseI footprint analysis of SarA binding to seqA and seqB fragments of P fmtA . A. Each DNA sequence was labeled with [γ- 32 P]ATP on the 5′-end of the bottom strand.

Techniques Used: Binding Assay, Sequencing, Labeling

Probing for fmtA transcription factor(s) in S. aureus cell extracts. (A) Binding reactions between 5′- 32 P-end labeled seq1, seq2 , and seq3 fragments of P fmtA and oxacillin-induced (+) or uninduced (-) S. aureus cell extracts. (B) EMSA of binding reactions between 5′-end [γ- 32 P] ATP labeled seq1 , seq2 and seq3 (2 ng) and oxacillin-induced S. aureus cell extracts in the presence and absence of unlabeled competitor (400 ng).
Figure Legend Snippet: Probing for fmtA transcription factor(s) in S. aureus cell extracts. (A) Binding reactions between 5′- 32 P-end labeled seq1, seq2 , and seq3 fragments of P fmtA and oxacillin-induced (+) or uninduced (-) S. aureus cell extracts. (B) EMSA of binding reactions between 5′-end [γ- 32 P] ATP labeled seq1 , seq2 and seq3 (2 ng) and oxacillin-induced S. aureus cell extracts in the presence and absence of unlabeled competitor (400 ng).

Techniques Used: Binding Assay, Labeling

65) Product Images from "Phosphorylation of p65(RelA) on Ser547 by ATM Represses NF-?B-Dependent Transcription of Specific Genes after Genotoxic Stress"

Article Title: Phosphorylation of p65(RelA) on Ser547 by ATM Represses NF-?B-Dependent Transcription of Specific Genes after Genotoxic Stress

Journal: PLoS ONE

doi: 10.1371/journal.pone.0038246

p65 interaction with ATM and p65 Ser 547 phosphorylation by ATM. ( A ) Confirmation of a direct interaction between ATM and p65 . Bacterially expressed GST or GST-ATM (1–247) fusion protein were purified on glutathione agarose beads and used to pull-down p65 from HEK-293 over-expressing HA-p65 cell lysate. Pulled down p65 was detected by immunoblotting with a p65 antibody (upper panel) and GST proteins were stained with coomassie bleu on the PVDF membrane. ( B ) Identification of the ATM target residue . Schematic representation of the different GST-p65 substrates used in the kinase assay. ( C ) In vitro kinase assay . Immunoprecipitated ATM from HEK-293 cells was incubated with GST-p53 and different GST-p65 proteins in presence of [γ− 32 P]ATP. The radiolabelled bands (upper panels) represent auto-phosphorylated ATM, phosphorylated GST-p53 or GST-p65. Levels of ATM and of substrate present in each reaction were determined by western blotting and by coomassie blue staining respectively (lower panels). ( D ) As in (C) In vitro kinase assay but with wt or mutated GST-p53 and GST-p65 proteins as substrates. ATM inhibitor KU55933 was added in some reaction samples as indicated. The same detection methodology than in ( C ) was used. ( E ) As in (D) in vitro kinase assay, but with purified recombinant ATM wt or kd instead of immunoprecipitated ATM and KU55933 utilization. ( F ) Conservation of Ser 547 among different species. Alignment of p65 C-terminal sequence from different mammalian and bird species.
Figure Legend Snippet: p65 interaction with ATM and p65 Ser 547 phosphorylation by ATM. ( A ) Confirmation of a direct interaction between ATM and p65 . Bacterially expressed GST or GST-ATM (1–247) fusion protein were purified on glutathione agarose beads and used to pull-down p65 from HEK-293 over-expressing HA-p65 cell lysate. Pulled down p65 was detected by immunoblotting with a p65 antibody (upper panel) and GST proteins were stained with coomassie bleu on the PVDF membrane. ( B ) Identification of the ATM target residue . Schematic representation of the different GST-p65 substrates used in the kinase assay. ( C ) In vitro kinase assay . Immunoprecipitated ATM from HEK-293 cells was incubated with GST-p53 and different GST-p65 proteins in presence of [γ− 32 P]ATP. The radiolabelled bands (upper panels) represent auto-phosphorylated ATM, phosphorylated GST-p53 or GST-p65. Levels of ATM and of substrate present in each reaction were determined by western blotting and by coomassie blue staining respectively (lower panels). ( D ) As in (C) In vitro kinase assay but with wt or mutated GST-p53 and GST-p65 proteins as substrates. ATM inhibitor KU55933 was added in some reaction samples as indicated. The same detection methodology than in ( C ) was used. ( E ) As in (D) in vitro kinase assay, but with purified recombinant ATM wt or kd instead of immunoprecipitated ATM and KU55933 utilization. ( F ) Conservation of Ser 547 among different species. Alignment of p65 C-terminal sequence from different mammalian and bird species.

Techniques Used: Purification, Expressing, Staining, Kinase Assay, In Vitro, Immunoprecipitation, Incubation, Western Blot, Recombinant, Sequencing

66) Product Images from "DNAzyme-mediated catalysis with only guanosine and cytidine nucleotides"

Article Title: DNAzyme-mediated catalysis with only guanosine and cytidine nucleotides

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkn930

Analysis of cleavage products. A 5′- 32 P-labelled substrate S4 was incubated with DNAzyme GC alone (lane 1) or in the presence of reaction buffer (lane 2). The reaction products were subsequently incubated in the presence of T4 PNK (lane 3) or T4 PNK and [γ- 32 P]-ATP (lanes 4 and 5). The products of lane 5 were incubated with 10-fold more [γ- 32 P]-ATP than lane 4. The 13 nt 5′-cleavage product (5′-P) and 8 nt 3′-cleavage product (3′-P) were separated from the uncleaved substrate by 20% denaturing PAGE. The reduced mobility of the 5′-cleavage product after treatment with PNK (lanes 3–5) is consistent with the removal of a 2′,3′-cyclic phosphate. The appearance of the 3′-cleavage product after treatment with PNK and [γ- 32 P]-ATP (lanes 4–5), is consistent with the phosphorylation of a free 5′-hydroxyl group.
Figure Legend Snippet: Analysis of cleavage products. A 5′- 32 P-labelled substrate S4 was incubated with DNAzyme GC alone (lane 1) or in the presence of reaction buffer (lane 2). The reaction products were subsequently incubated in the presence of T4 PNK (lane 3) or T4 PNK and [γ- 32 P]-ATP (lanes 4 and 5). The products of lane 5 were incubated with 10-fold more [γ- 32 P]-ATP than lane 4. The 13 nt 5′-cleavage product (5′-P) and 8 nt 3′-cleavage product (3′-P) were separated from the uncleaved substrate by 20% denaturing PAGE. The reduced mobility of the 5′-cleavage product after treatment with PNK (lanes 3–5) is consistent with the removal of a 2′,3′-cyclic phosphate. The appearance of the 3′-cleavage product after treatment with PNK and [γ- 32 P]-ATP (lanes 4–5), is consistent with the phosphorylation of a free 5′-hydroxyl group.

Techniques Used: Incubation, Polyacrylamide Gel Electrophoresis

67) Product Images from "Essential role of the Cdk2 activator RingoA in meiotic telomere tethering to the nuclear envelope"

Article Title: Essential role of the Cdk2 activator RingoA in meiotic telomere tethering to the nuclear envelope

Journal: Nature Communications

doi: 10.1038/ncomms11084

RingoA interacts with Cdk2 and regulates Cdk2 kinase activity in testis. ( a ) Immunoprecipitates from WT testis lysates using RingoA or Cdk2 antibodies and control IgGs were analysed by immunoblotting for RingoA. The arrowhead indicates RingoA and the asterisks IgGs. ( b ) WT and KO 18 dpp old testis lysates were immunoprecipitated with Cdk2 antibodies or control IgGs. The immunoprecipitates were assayed in an in vitro kinase assay using γ- 32 P-ATP and Rb protein as a substrate (upper row) and the gel was stained with Coomassie (middle row). An aliquot of the immunoprecipitates was analysed by immunoblotting with Cdk2 antibodies (bottom row). The phosphorylated Rb protein was quantified using ImageJ and was referred to the amount of RB protein detected in the Coomassie staining. The experiments were reproduced two to three times. The uncropped images are shown in Supplementary Fig. 7 .
Figure Legend Snippet: RingoA interacts with Cdk2 and regulates Cdk2 kinase activity in testis. ( a ) Immunoprecipitates from WT testis lysates using RingoA or Cdk2 antibodies and control IgGs were analysed by immunoblotting for RingoA. The arrowhead indicates RingoA and the asterisks IgGs. ( b ) WT and KO 18 dpp old testis lysates were immunoprecipitated with Cdk2 antibodies or control IgGs. The immunoprecipitates were assayed in an in vitro kinase assay using γ- 32 P-ATP and Rb protein as a substrate (upper row) and the gel was stained with Coomassie (middle row). An aliquot of the immunoprecipitates was analysed by immunoblotting with Cdk2 antibodies (bottom row). The phosphorylated Rb protein was quantified using ImageJ and was referred to the amount of RB protein detected in the Coomassie staining. The experiments were reproduced two to three times. The uncropped images are shown in Supplementary Fig. 7 .

Techniques Used: Activity Assay, Immunoprecipitation, In Vitro, Kinase Assay, Staining

RingoA regulates Sun1 telomeric localization. ( a ) WT and KO spread spermatocytes were immunolabelled for Sycp3 (red) and Terb1 (green). Terb1 signals at telomeres were unperturbed in RingoA KO spermatocytes. ( b ) WT and KO spread spermatocytes were immunolabeled for Sycp3 (red) and Sun1 (green). Telomeric localization of Sun1 was abolished in KO spermatocytes. Scale bar, 10 μm. ( c ) Purified MBP-fused proteins including amino acids 1-217 (either WT or with the mutation S48A) or amino acids 456–916 of Sun1 were incubated with Cdk2 and RingoA in the presence of [γ- 32 P]ATP. Proteins were analysed by SDS–PAGE, stained with Coomassie Brilliant Blue (CBB) and subjected to autoradiography ( 32 P). The experiments were reproduced three times. The uncropped autoradiography is shown in Supplementary Fig. 7 .
Figure Legend Snippet: RingoA regulates Sun1 telomeric localization. ( a ) WT and KO spread spermatocytes were immunolabelled for Sycp3 (red) and Terb1 (green). Terb1 signals at telomeres were unperturbed in RingoA KO spermatocytes. ( b ) WT and KO spread spermatocytes were immunolabeled for Sycp3 (red) and Sun1 (green). Telomeric localization of Sun1 was abolished in KO spermatocytes. Scale bar, 10 μm. ( c ) Purified MBP-fused proteins including amino acids 1-217 (either WT or with the mutation S48A) or amino acids 456–916 of Sun1 were incubated with Cdk2 and RingoA in the presence of [γ- 32 P]ATP. Proteins were analysed by SDS–PAGE, stained with Coomassie Brilliant Blue (CBB) and subjected to autoradiography ( 32 P). The experiments were reproduced three times. The uncropped autoradiography is shown in Supplementary Fig. 7 .

Techniques Used: Immunolabeling, Purification, Mutagenesis, Incubation, SDS Page, Staining, Autoradiography

68) Product Images from "L290P/V mutations increase ERK3’s cytoplasmic localization and migration/invasion-promoting capability in cancer cells"

Article Title: L290P/V mutations increase ERK3’s cytoplasmic localization and migration/invasion-promoting capability in cancer cells

Journal: Scientific Reports

doi: 10.1038/s41598-017-15135-9

L290P/V mutations do not alter ERK3 kinase activity. ( a ) Coomassie staining of purified wild type or mutant ERK3 proteins. 293 T cells were transfected with HA-tagged wild type ERK3, ERK3 L290P, L290V or kinase dead (KD) plasmids. ERK3 proteins were immunoprecipitated using HA-antibody-conjugated agarose beads, followed by elution with HA peptide. The purified proteins (300 ng) were analyzed by SDS-PAGE gel followed by Coomassie staining. The molecular size of each protein marker is indicated on the right side. ( b ) In vitro ERK3 kinase assay was performed by incubating 100 ng of purified ERK3 or each of ERK3 mutants as indicated, together with 1 μg of recombinant GST-SRC3-CID (substrate) in the presence of γ- 32 P-ATP. Phosphorylation of GST-SRC3-CID by ERK3 proteins was detected by autoradiograph (the right panel). Total protein level of GST-SRC3-CID in the reactions is shown by Coomassie staining (the left panel). Please note that ERK3 proteins are hardly seen in the Coomassie-stained gel due to their small amount (100 ng). ( c ) Quantification of GST-SRC3-CID phosphorylation by wild type or mutant ERK3 proteins. The relative phosphorylation level of GST-SRC3-CID is represented by the ratio of the band intensity of phosphorylated GST-SRC3-CID (shown in the autoradiograph) over that of the corresponding total GST-SRC3-CID (shown in the commassie-stained gel). For the purpose of comparison, the nomalized phosphorylation level of GST-SRC3-CID by wild type ERK3 was arbitrarily set as 1.0. The bar graph represents the mean ± S.E. of 3 independent experiments. *P
Figure Legend Snippet: L290P/V mutations do not alter ERK3 kinase activity. ( a ) Coomassie staining of purified wild type or mutant ERK3 proteins. 293 T cells were transfected with HA-tagged wild type ERK3, ERK3 L290P, L290V or kinase dead (KD) plasmids. ERK3 proteins were immunoprecipitated using HA-antibody-conjugated agarose beads, followed by elution with HA peptide. The purified proteins (300 ng) were analyzed by SDS-PAGE gel followed by Coomassie staining. The molecular size of each protein marker is indicated on the right side. ( b ) In vitro ERK3 kinase assay was performed by incubating 100 ng of purified ERK3 or each of ERK3 mutants as indicated, together with 1 μg of recombinant GST-SRC3-CID (substrate) in the presence of γ- 32 P-ATP. Phosphorylation of GST-SRC3-CID by ERK3 proteins was detected by autoradiograph (the right panel). Total protein level of GST-SRC3-CID in the reactions is shown by Coomassie staining (the left panel). Please note that ERK3 proteins are hardly seen in the Coomassie-stained gel due to their small amount (100 ng). ( c ) Quantification of GST-SRC3-CID phosphorylation by wild type or mutant ERK3 proteins. The relative phosphorylation level of GST-SRC3-CID is represented by the ratio of the band intensity of phosphorylated GST-SRC3-CID (shown in the autoradiograph) over that of the corresponding total GST-SRC3-CID (shown in the commassie-stained gel). For the purpose of comparison, the nomalized phosphorylation level of GST-SRC3-CID by wild type ERK3 was arbitrarily set as 1.0. The bar graph represents the mean ± S.E. of 3 independent experiments. *P

Techniques Used: Activity Assay, Staining, Purification, Mutagenesis, Transfection, Immunoprecipitation, SDS Page, Marker, In Vitro, Kinase Assay, Recombinant, Autoradiography

69) 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

70) Product Images from "Small molecules that allosterically inhibit p21-activated kinase activity by binding to the regulatory p21-binding domain"

Article Title: Small molecules that allosterically inhibit p21-activated kinase activity by binding to the regulatory p21-binding domain

Journal: Experimental & Molecular Medicine

doi: 10.1038/emm.2016.13

Suppression of PAK1 activity by p21-binding domain (PBD) binders in vitro . ( a ) Inhibition of Cdc42-dependent PAK1 activation. Cdc42•GTP, Wt-PAK1, myelin basic protein (MBP) and [γ- 32 P]-ATP were incubated with various concentrations of the indicated compounds for 0.5 h. Immunoblotting of total MBP was performed as a loading control. ( b ) Direct inhibition of PAK1 activity by compounds in vitro . Active PAK1 T423E , MBP and [γ- 32 P]-ATP were incubated with various concentrations of the indicated compounds for 0.5 h. Immunoblotting of total MBP was performed as a loading control. ( c ) Full-length PAK1 T423E/LL or PAK1 T423E was incubated with MBP and [γ- 32 P]-ATP in the presence of dimethylsulphoxide (control) or the indicated compounds (40 μ M ) for 0.5 h. LL, H83L/H86L. ( d ) Activity of full-length PAK1 T423E or a single-substitution mutant was measured in the absence or presence of compounds as described in c . In all experiments, MBP phosphorylation was analyzed by autoradiography. All data are representative of at least three independent experiments.
Figure Legend Snippet: Suppression of PAK1 activity by p21-binding domain (PBD) binders in vitro . ( a ) Inhibition of Cdc42-dependent PAK1 activation. Cdc42•GTP, Wt-PAK1, myelin basic protein (MBP) and [γ- 32 P]-ATP were incubated with various concentrations of the indicated compounds for 0.5 h. Immunoblotting of total MBP was performed as a loading control. ( b ) Direct inhibition of PAK1 activity by compounds in vitro . Active PAK1 T423E , MBP and [γ- 32 P]-ATP were incubated with various concentrations of the indicated compounds for 0.5 h. Immunoblotting of total MBP was performed as a loading control. ( c ) Full-length PAK1 T423E/LL or PAK1 T423E was incubated with MBP and [γ- 32 P]-ATP in the presence of dimethylsulphoxide (control) or the indicated compounds (40 μ M ) for 0.5 h. LL, H83L/H86L. ( d ) Activity of full-length PAK1 T423E or a single-substitution mutant was measured in the absence or presence of compounds as described in c . In all experiments, MBP phosphorylation was analyzed by autoradiography. All data are representative of at least three independent experiments.

Techniques Used: Activity Assay, Binding Assay, In Vitro, Inhibition, Activation Assay, Incubation, Mutagenesis, Autoradiography

71) Product Images from "Bacteriophage T7 protein kinase. Site of inhibitory autophosphorylation, and use of dephosphorylated enzyme for efficient modification of protein in vitro"

Article Title: Bacteriophage T7 protein kinase. Site of inhibitory autophosphorylation, and use of dephosphorylated enzyme for efficient modification of protein in vitro

Journal: Protein expression and purification

doi: 10.1016/j.pep.2012.08.008

T7PK phosphorylation of IF1 and the RNase III Nuclease Domain (NucD). Purified E. coli IF1 or E. coli RNase III NucD (both lacking the His-Tag) was phosphorylated in the presence of [γ- 32 P]ATP using H-pT7PK, H-T7PK, or T7PK as described in Materials and Methods . Aliquots were fractionated by 12% SDS-PAGE and visualized by phosphorimaging as described in Materials and Methods . A. T7PK phosphorylation of IF1. Lane 1: Phosphorylation reaction lacking IF1. Lane 2: IF1 phosphorylated by H-pT7PK. Lane 3: IF1 phosphorylated by H-T7PK. Lane 4: IF1 phosphorylated by T7PK. B. Phosphorylation of RNase III NucD. Lane 1: Phosphorylation reaction lacking NucD. Lane 2: NucD phosphorylated by H-pT7PK. Lane 3: NucD phosphorylated by H-T7PK. Lane 4: NucD phosphorylation by T7PK.
Figure Legend Snippet: T7PK phosphorylation of IF1 and the RNase III Nuclease Domain (NucD). Purified E. coli IF1 or E. coli RNase III NucD (both lacking the His-Tag) was phosphorylated in the presence of [γ- 32 P]ATP using H-pT7PK, H-T7PK, or T7PK as described in Materials and Methods . Aliquots were fractionated by 12% SDS-PAGE and visualized by phosphorimaging as described in Materials and Methods . A. T7PK phosphorylation of IF1. Lane 1: Phosphorylation reaction lacking IF1. Lane 2: IF1 phosphorylated by H-pT7PK. Lane 3: IF1 phosphorylated by H-T7PK. Lane 4: IF1 phosphorylated by T7PK. B. Phosphorylation of RNase III NucD. Lane 1: Phosphorylation reaction lacking NucD. Lane 2: NucD phosphorylated by H-pT7PK. Lane 3: NucD phosphorylated by H-T7PK. Lane 4: NucD phosphorylation by T7PK.

Techniques Used: Purification, SDS Page

Purification and dephosphorylation of T7PK. A. Analysis of purified H-pT7PK by 12% SDS-PAGE and Coomassie staining. Lane 1: protein standards (sizes in kDa). Lane 2. Purified H-pT7PK, with an apparent molecular mass of 30 kDa. B. Phosphorimage of T7PK autophosphorylated in the presence of [γ- 32 P]ATP, then analyzed by 12% SDS-PAGE as described in Materials and Methods . C. Electrophoretic mobility differences of H-pT7PK, H-T7PK, and T7PK. Shown is an image of a Coomassie-stained, 15% polyacrylamide-SDS gel. Lane 1: protein standards (sizes in kDa). Lane 2: H-pT7PK as directly purified from cells. Lane 3: H-T7PK, obtained by lambda phage protein phosphatase treatment of H-pT7PK. Lane 4: T7PK obtained by biotinylated thrombin treatment of H-T7PK. Note the greater mobility of T7PK compared to His-pT7PK or His-T7PK.
Figure Legend Snippet: Purification and dephosphorylation of T7PK. A. Analysis of purified H-pT7PK by 12% SDS-PAGE and Coomassie staining. Lane 1: protein standards (sizes in kDa). Lane 2. Purified H-pT7PK, with an apparent molecular mass of 30 kDa. B. Phosphorimage of T7PK autophosphorylated in the presence of [γ- 32 P]ATP, then analyzed by 12% SDS-PAGE as described in Materials and Methods . C. Electrophoretic mobility differences of H-pT7PK, H-T7PK, and T7PK. Shown is an image of a Coomassie-stained, 15% polyacrylamide-SDS gel. Lane 1: protein standards (sizes in kDa). Lane 2: H-pT7PK as directly purified from cells. Lane 3: H-T7PK, obtained by lambda phage protein phosphatase treatment of H-pT7PK. Lane 4: T7PK obtained by biotinylated thrombin treatment of H-T7PK. Note the greater mobility of T7PK compared to His-pT7PK or His-T7PK.

Techniques Used: Purification, De-Phosphorylation Assay, SDS Page, Staining, SDS-Gel

72) Product Images from "Mechanism of ATP turnover inhibition in the EJC"

Article Title: Mechanism of ATP turnover inhibition in the EJC

Journal: RNA

doi: 10.1261/rna.1283109

ATP hydrolysis can occur in the EJC. Thin-layer chromatography of ATP-α 32 P (lane 1 ), ATP-α 32 P treated with recombinant human Upf1 showing the position of ADP-α 32 P (lane 2 ) and ATP-γ 32 P treated with recombinant human Upf1
Figure Legend Snippet: ATP hydrolysis can occur in the EJC. Thin-layer chromatography of ATP-α 32 P (lane 1 ), ATP-α 32 P treated with recombinant human Upf1 showing the position of ADP-α 32 P (lane 2 ) and ATP-γ 32 P treated with recombinant human Upf1

Techniques Used: Thin Layer Chromatography, Recombinant

73) Product Images from "Trypanosoma brucei Vacuolar Transporter Chaperone 4 (TbVtc4) Is an Acidocalcisome Polyphosphate Kinase Required for in Vivo Infection *"

Article Title: Trypanosoma brucei Vacuolar Transporter Chaperone 4 (TbVtc4) Is an Acidocalcisome Polyphosphate Kinase Required for in Vivo Infection *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M113.518993

TBE-PAGE analysis of polyP produced by TbVtc4 and ScVtc4p catalytic cores. PolyP synthesized by recombinant TbVtc4 and ScVtc4p using [γ- 32 P]ATP as substrate was analyzed by TBE-PAGE. A , autoradiography of TbVtc4 reaction products in the presence
Figure Legend Snippet: TBE-PAGE analysis of polyP produced by TbVtc4 and ScVtc4p catalytic cores. PolyP synthesized by recombinant TbVtc4 and ScVtc4p using [γ- 32 P]ATP as substrate was analyzed by TBE-PAGE. A , autoradiography of TbVtc4 reaction products in the presence

Techniques Used: Polyacrylamide Gel Electrophoresis, Produced, Synthesized, Recombinant, Autoradiography

74) Product Images from "Activation of yeast Snf1 and mammalian AMP-activated protein kinase by upstream kinases"

Article Title: Activation of yeast Snf1 and mammalian AMP-activated protein kinase by upstream kinases

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

doi: 10.1073/pnas.1533136100

Tos3p, Pak1p, and Elm1p phosphorylate Snf1p on T210 in vitro ) were purified from yeast cells. The GST-kinase, immobilized on beads, was incubated with bacterially expressed Snf1KD-K84R or -T210A (0.2 μg), or no substrate, in the presence of [γ- 32 P]ATP for 20 min at 25°C. Proteins were analyzed by SDS/PAGE and autoradiography. An autoradiogram is shown. An arbitrary GST-kinase, YPL141C, served as a control. Arrowheads, mutant Snf1KD; asterisks, autophosphorylated full-length GST-kinase. Molecular mass markers (kDa) are indicated.
Figure Legend Snippet: Tos3p, Pak1p, and Elm1p phosphorylate Snf1p on T210 in vitro ) were purified from yeast cells. The GST-kinase, immobilized on beads, was incubated with bacterially expressed Snf1KD-K84R or -T210A (0.2 μg), or no substrate, in the presence of [γ- 32 P]ATP for 20 min at 25°C. Proteins were analyzed by SDS/PAGE and autoradiography. An autoradiogram is shown. An arbitrary GST-kinase, YPL141C, served as a control. Arrowheads, mutant Snf1KD; asterisks, autophosphorylated full-length GST-kinase. Molecular mass markers (kDa) are indicated.

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

Assays of Snf1 kinase activity. ( A and B )] were grown in selective SC plus 2% glucose. Proteins were immunoprecipitated from extracts (200 μg) with anti-LexA. ( A ) Immunoprecipitates were incubated in kinase buffer containing [γ- 32 P]ATP and analyzed by SDS/PAGE and autoradiography. ( B ) Immunoprecipitates were analyzed by immunoblotting with anti-LexA. ( C ). A snf1-K84R mutant extract also showed no activity. ( D ) The assayed fractions were immunoblotted with anti-Snf1.
Figure Legend Snippet: Assays of Snf1 kinase activity. ( A and B )] were grown in selective SC plus 2% glucose. Proteins were immunoprecipitated from extracts (200 μg) with anti-LexA. ( A ) Immunoprecipitates were incubated in kinase buffer containing [γ- 32 P]ATP and analyzed by SDS/PAGE and autoradiography. ( B ) Immunoprecipitates were analyzed by immunoblotting with anti-LexA. ( C ). A snf1-K84R mutant extract also showed no activity. ( D ) The assayed fractions were immunoblotted with anti-Snf1.

Techniques Used: Activity Assay, Immunoprecipitation, Incubation, SDS Page, Autoradiography, Mutagenesis

Tos3p and LKB1 phosphorylate and activate AMPK in vitro .( A ). ( B ) A catalytically inactive mutant AMPK (D157A substitution) was incubated with GST-Tos3p or GST bound to glutathione-Sepharose beads in the presence of [γ- 32 P]ATP, and proteins were analyzed by SDS/PAGE and autoradiography. Molecular mass markers (kDa) are indicated. ( C ) and analyzed by immunoblotting with antibody specific to phosphothreonine 172. ( D and E ) Purified, immobilized FLAG-tagged LKB1, either WT or a catalytically inactive mutant (D194A), was incubated with bacterially expressed AMPK. The empty vector is pCDNA3 (Invitrogen) lacking LKB1 insert. ( D ). ( E ) Phosphorylation of T172 was determined by immunoblotting as in C .
Figure Legend Snippet: Tos3p and LKB1 phosphorylate and activate AMPK in vitro .( A ). ( B ) A catalytically inactive mutant AMPK (D157A substitution) was incubated with GST-Tos3p or GST bound to glutathione-Sepharose beads in the presence of [γ- 32 P]ATP, and proteins were analyzed by SDS/PAGE and autoradiography. Molecular mass markers (kDa) are indicated. ( C ) and analyzed by immunoblotting with antibody specific to phosphothreonine 172. ( D and E ) Purified, immobilized FLAG-tagged LKB1, either WT or a catalytically inactive mutant (D194A), was incubated with bacterially expressed AMPK. The empty vector is pCDNA3 (Invitrogen) lacking LKB1 insert. ( D ). ( E ) Phosphorylation of T172 was determined by immunoblotting as in C .

Techniques Used: In Vitro, Mutagenesis, Incubation, SDS Page, Autoradiography, Purification, Plasmid Preparation

75) Product Images from "Cyclin-dependent kinase 5 mediates pleiotrophin-induced endothelial cell migration"

Article Title: Cyclin-dependent kinase 5 mediates pleiotrophin-induced endothelial cell migration

Journal: Scientific Reports

doi: 10.1038/s41598-018-24326-x

PTN enhances CDK5 activity. ( a ) Effect of PTN (100 ng/ml) on CDK5 activity as assessed by histone H1 phosphorylation in vitro . Total HUVEC lysates were immunoprecipitated with a CDK5 antibody and immunoprecipitates were incubated with histone Η1 (20 μg) and [γ- 32 P] ATP (20 μCi) for 30 min at 30 °C, as described in Materials and Methods. A representative autoradiography from three independent experiments is presented. The bands corresponding to phosphorylated histone Η1 were quantified by image analysis software and normalized against total CDK5 levels in each sample. Results are expressed as mean ± SE (n = 3) of the percent change of histone H1 phosphorylation compared to untreated cells (set as default 100%). Asterisks denote statistically significant differences from the untreated cells; **P
Figure Legend Snippet: PTN enhances CDK5 activity. ( a ) Effect of PTN (100 ng/ml) on CDK5 activity as assessed by histone H1 phosphorylation in vitro . Total HUVEC lysates were immunoprecipitated with a CDK5 antibody and immunoprecipitates were incubated with histone Η1 (20 μg) and [γ- 32 P] ATP (20 μCi) for 30 min at 30 °C, as described in Materials and Methods. A representative autoradiography from three independent experiments is presented. The bands corresponding to phosphorylated histone Η1 were quantified by image analysis software and normalized against total CDK5 levels in each sample. Results are expressed as mean ± SE (n = 3) of the percent change of histone H1 phosphorylation compared to untreated cells (set as default 100%). Asterisks denote statistically significant differences from the untreated cells; **P

Techniques Used: Activity Assay, In Vitro, Immunoprecipitation, Incubation, Autoradiography, Software

Effect of the studied pyrrolo[2,3-α]carbazole derivatives on PTN-induced CDK5 activation and cell migration in vitro . ( a ) HUVEC were treated with the tested agents (10 μΜ) for 30 min, prior to stimulation with PTN (100 ng/ml) for 5 min. Whole cell lysates were immunoprecipitated for CDK5 and immunoprecipitates were incubated with histone Η1 (20 μg) and [γ- 32 P] ATP (20 μCi) for 30 min at 30 °C, as described in Materials and Methods. A representative autoradiography from three independent experiments is presented. The bands corresponding to phosphorylated histone Η1 were quantified by image analysis software and were normalized against total CDK5 levels in each sample. Results are expressed as mean ± SE (n = 3) of the percent change of histone H1 phosphorylation compared to untreated cells (set as default 100%). ( b ) Serum-starved HUVEC were stimulated by PTN (100 ng/ml) in the absence or presence of the tested agents (10 μΜ). Migration was measured using the transwell assay, as described in Materials and Methods. Results are expressed as mean ± SE (n = 3) of the percentage change compared to the corresponding untreated cells (set as default 100%). Asterisks denote statistically significant differences from the untreated cells; *P
Figure Legend Snippet: Effect of the studied pyrrolo[2,3-α]carbazole derivatives on PTN-induced CDK5 activation and cell migration in vitro . ( a ) HUVEC were treated with the tested agents (10 μΜ) for 30 min, prior to stimulation with PTN (100 ng/ml) for 5 min. Whole cell lysates were immunoprecipitated for CDK5 and immunoprecipitates were incubated with histone Η1 (20 μg) and [γ- 32 P] ATP (20 μCi) for 30 min at 30 °C, as described in Materials and Methods. A representative autoradiography from three independent experiments is presented. The bands corresponding to phosphorylated histone Η1 were quantified by image analysis software and were normalized against total CDK5 levels in each sample. Results are expressed as mean ± SE (n = 3) of the percent change of histone H1 phosphorylation compared to untreated cells (set as default 100%). ( b ) Serum-starved HUVEC were stimulated by PTN (100 ng/ml) in the absence or presence of the tested agents (10 μΜ). Migration was measured using the transwell assay, as described in Materials and Methods. Results are expressed as mean ± SE (n = 3) of the percentage change compared to the corresponding untreated cells (set as default 100%). Asterisks denote statistically significant differences from the untreated cells; *P

Techniques Used: Activation Assay, Migration, In Vitro, Immunoprecipitation, Incubation, Autoradiography, Software, Transwell Assay

Related Articles

Centrifugation:

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
Article Snippet: Lysates were cleared by centrifugation before addition of T1 RNase (final concentration of 0.025 U/µL, cat. no. EN0541, Fermentas, St. Leon-Rot, Germany) and incubation for 6 minutes at 37°C with 1,000-rpm agitation. .. The immunoprecipitated samples were dephosphorylated by calf intestinal phosphatase (final concentration of 0.038 U/µL, cat. no. 10713023001, Roche Diagnostics) for 10 minutes and washed 3 times with Buffer C (50 mM Tris HCl, pH 7,4, 10 mM MgCl2 , 0.5% NP-40, 1x protease inhibitor cocktail [cat. no. P8340, Sigma-Aldrich]), followed by labeling with γ-[32 P]-ATP (2.1 µCi, cat. no. NEG502H, Perkin Elmer, Maanstraat, Germany) by polynucleotide kinase phosphorylation (0.5 U/µL, cat. no. M0201L, New England Biolabs Inc., Ipswich, MA) and further washed 3 times with Buffer C. Samples were resolved on 6% denaturing polyacrylamide gels.

Article Title: Eukaryotic Elongation Factor 1A Interacts with Sphingosine Kinase and Directly Enhances Its Catalytic Activity *
Article Snippet: After this time the GSH-Sepharose beads (with bound protein) were pelleted by centrifugation at 3000 × g for 5 min at 4 °C and washed twice in cold PBS. .. Sphingosine Kinase Assays —Sphingosine kinase activity was routinely determined using d - erythro -sphingosine (Biomol, Plymouth Meeting, PA) and γ-32 P]ATP (PerkinElmer Life Sciences) as substrates, as described previously ( ).

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: Molecular mechanisms of two-component system RhpRS regulating type III secretion system in Pseudomonas syringae
Article Snippet: Production of [γ-32 P]-acetyl phosphate that phosphorylates RhpR [γ-32 P]-acetyl phosphate was synthesized as described ( , ). .. Briefly, the reaction mixture including 10-μM putative acetate kinase PSPTO_1669 (AckA), 10 μCi of [γ-32 P]-ATP (6000 Ci/mmol, PerkinElmer) in an acetate kinase buffer (25-mM Tris–HCl [pH 7.4], 60-mM KOAc, 10-mM MgCl2 ) was incubated at room temperature for 30 min before removing acetate kinase by using a 30 kDa cut-off column (Amicon ultra with 30 kDa cut-off, Millipore).

Article Title: Discovery of catalytically active orthologues of the Parkinson's disease kinase PINK1: analysis of substrate specificity and impact of mutations
Article Snippet: [γ-32 P] ATP was from PerkinElmer (Waltham, MA). .. PINKtide and its derivatives were synthesized by GL Biochem (Shanghai, China).

Autoradiography:

Article Title: Metabolism of circulating ADP in the bloodstream is mediated via integrated actions of soluble adenylate kinase-1 and NTPDase1/CD39 activities
Article Snippet: 3 H-labeled nucleotides and nucleosides were separated by TLC and then either quantified by scintillation β-counting ( ) or exposed to Kodak BioMax MS films (Eastman Kodak, Rochester, NY, USA) for 2 wk at −70°C and developed by autoradiography. .. Autoradiographic analysis was also performed by incubating 4 μl murine serum for 30 min in 60 μl RPMI 1640 containing 4 mM β-glycerophosphate, 50 μM [γ-32 P]ATP (Perkin Elmer) and various unlabeled nucleotides, followed by TLC separation of mixture aliquots on Polygram CEL-300 PEI sheets (Macherey-Nagel) with 0.75 M KH2 PO4 (pH 3.5) as solvent.

Article Title: DNA-PK Target Identification Reveals Novel Links between DNA Repair Signaling and Cytoskeletal Regulation
Article Snippet: Paragraph title: In vitro phosphorylation and autoradiography ... We incubated 0–3.5 µg vimentin or 0–10 µg microtubules with 200 U purified DNA-PK (Promega, Lyons, France), 0.1 mM ATP, 0.001 mM (γ-32 P)ATP (10 mCi/ml, Perkin Elmer, Courtaboeuf, France), 10 µg/ml bovine serum albumin (BSA) and 0.1 µg/µl Dbait32Hc or 0.03 µg/µl thymus DNA (Promega) in 20 mM KCl, 0.2 mM DTT, 10 mM HEPES/KOH pH 7.5, 2 mM MgCl2 , 0.04 mM EGTA, 0.02 mM EDTA/KOH pH 7.5.

Incubation:

Article Title: Extracellular Signal-Regulated Kinase Promotes Rho-Dependent Focal Adhesion Formation by Suppressing p190A RhoGAP ▿
Article Snippet: After phosphatase treatment, immunoprecipitates were washed in kinase buffer (20 mM Tris, pH 7.8, 10 mM MgCl2 ,) and suspended in 50 μl of kinase buffer supplemented with 1 mM DTT, 10 μM ATP, 10 μCi [γ-32 P]ATP (EazyTide; Perkin Elmer) and 0 to 100 ng of recombinant ERK1 (MBL International Corporation). .. After 30 min of incubation at 30°C, kinase reactions were stopped by adding 2× SDS-PAGE sample buffer and analyzed by PhosphorImager or exposure to X-ray film.

Article Title: Active site–adjacent phosphorylation at Tyr-397 by c-Abl kinase inactivates caspase-9
Article Snippet: .. c-Abl (20 μ m ) was incubated in kinase activity buffer (50 m m Tris-Cl, pH 7.5, 20 m m MgCl2 , 0.1 m m EDTA, 0.5 m m EGTA, 5 m m β-glycerophosphate, 1 m m Na3 VO4 ) and allowed to autoactivate in the presence of 250 μ m ATP with [γ-32 P]ATP (10 μCi/μl; PerkinElmer Life Sciences) for 2 h at 30 °C. .. Casp-9 (50 μ m ) was incubated with 1 μ m autoactivated c-Abl in kinase activity buffer with 1 m m ATP containing [γ-32 P]ATP for 4 h at 30 °C.

Article Title: Molecular mechanisms of two-component system RhpRS regulating type III secretion system in Pseudomonas syringae
Article Snippet: .. Briefly, the reaction mixture including 10-μM putative acetate kinase PSPTO_1669 (AckA), 10 μCi of [γ-32 P]-ATP (6000 Ci/mmol, PerkinElmer) in an acetate kinase buffer (25-mM Tris–HCl [pH 7.4], 60-mM KOAc, 10-mM MgCl2 ) was incubated at room temperature for 30 min before removing acetate kinase by using a 30 kDa cut-off column (Amicon ultra with 30 kDa cut-off, Millipore). ..

Article Title: Metabolism of circulating ADP in the bloodstream is mediated via integrated actions of soluble adenylate kinase-1 and NTPDase1/CD39 activities
Article Snippet: In addition, human AK was determined after 45 min incubation of serum or plasma (10 μl) with 400 μM [3 H]AMP plus 750 μM ATP, while for ADPase assays, blood samples were incubated for 60 min with 50 μM [3 H]ADP in the presence of 100 μM Ap5 A. .. Autoradiographic analysis was also performed by incubating 4 μl murine serum for 30 min in 60 μl RPMI 1640 containing 4 mM β-glycerophosphate, 50 μM [γ-32 P]ATP (Perkin Elmer) and various unlabeled nucleotides, followed by TLC separation of mixture aliquots on Polygram CEL-300 PEI sheets (Macherey-Nagel) with 0.75 M KH2 PO4 (pH 3.5) as solvent.

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
Article Snippet: .. DNA kinase assays PNKP (10 pmol) was premixed with 40 pmol of full-length XRCC1-His, His-XRCC1FFF , His-XRCC1161–406 , or His-XRCC1161–406 R335A/K369A at 37 °C for 5 min and then the mixtures were added to 20-μl (total volume) reactions containing kinase buffer (80 mm succinic acid, pH 5.5, 10 mm MgCl2 , and 1 mm dithiothreitol), 0.2 nmol of 24-mer 5′-DNA kinase substrate (Integrated DNA Technologies; the single- and double-stranded DNA substrates used in this study have been described previously ( )) and 3.3 pmol of [γ-32 P]ATP (PerkinElmer Life Sciences) and incubated for 2 min at 37 °C. .. 4-μl aliquots were mixed with 2 μl of 3× sequencing gel loading dye (Fisher), boiled for 10 min, and fractionated on a 12% polyacrylamide, 7 m urea sequencing gel at 200 V. Gels were imaged on a Typhoon 9400 variable mode imager (GE Healthcare, Bucks, UK) and quantified using ImageQuant 5.2 software (GE Healthcare).

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
Article Snippet: Lysates were cleared by centrifugation before addition of T1 RNase (final concentration of 0.025 U/µL, cat. no. EN0541, Fermentas, St. Leon-Rot, Germany) and incubation for 6 minutes at 37°C with 1,000-rpm agitation. .. The immunoprecipitated samples were dephosphorylated by calf intestinal phosphatase (final concentration of 0.038 U/µL, cat. no. 10713023001, Roche Diagnostics) for 10 minutes and washed 3 times with Buffer C (50 mM Tris HCl, pH 7,4, 10 mM MgCl2 , 0.5% NP-40, 1x protease inhibitor cocktail [cat. no. P8340, Sigma-Aldrich]), followed by labeling with γ-[32 P]-ATP (2.1 µCi, cat. no. NEG502H, Perkin Elmer, Maanstraat, Germany) by polynucleotide kinase phosphorylation (0.5 U/µL, cat. no. M0201L, New England Biolabs Inc., Ipswich, MA) and further washed 3 times with Buffer C. Samples were resolved on 6% denaturing polyacrylamide gels.

Article Title: Transcriptional Corepressors HIPK1 and HIPK2 Control Angiogenesis Via TGF-?-TAK1-Dependent Mechanism
Article Snippet: .. Immune complexes were washed with kinase buffer (25 mM Tris-HCl, pH 8.0, 10 mM MgCl2 ), and then incubated with 1 mM ATP and 5 µCi of γ-32 P-ATP (Perkin Elmer) for 3 h at room temperature. ..

Article Title: Preformed Soluble Chemoreceptor Trimers That Mimic Cellular Assembly States and Activate CheA Autophosphorylation
Article Snippet: .. After incubation, 2 μL of 2.3 mM cold ATP and 3–8 μL of a [γ-32 P]ATP (3000 Ci/mmol, 10 mCi/mL, PerkinElmer) solution were added to the sample to produce a total volume of 25 μL. .. After [γ-32 P]ATP exposure times from 10 s to 12 min (up to 40 min for TarFO short), the sample was quenched with 25 μL of 3× SDS with 50 mM EDTA (pH 8.0) and then subjected to gel electrophoresis on a 4–20% gradient Tris-glycine gel.

Article Title: DNA-PK Target Identification Reveals Novel Links between DNA Repair Signaling and Cytoskeletal Regulation
Article Snippet: .. We incubated 0–3.5 µg vimentin or 0–10 µg microtubules with 200 U purified DNA-PK (Promega, Lyons, France), 0.1 mM ATP, 0.001 mM (γ-32 P)ATP (10 mCi/ml, Perkin Elmer, Courtaboeuf, France), 10 µg/ml bovine serum albumin (BSA) and 0.1 µg/µl Dbait32Hc or 0.03 µg/µl thymus DNA (Promega) in 20 mM KCl, 0.2 mM DTT, 10 mM HEPES/KOH pH 7.5, 2 mM MgCl2 , 0.04 mM EGTA, 0.02 mM EDTA/KOH pH 7.5. .. The mixture was incubated for 30 min at 30°C and the reaction was stopped by adding 2×SDS sample buffer to give a final concentration of 50 mM Tris-HCl (pH 6.8), 1% β-mercaptoethanol, 2% SDS, 0.1% bromophenol blue and 10% glycerol.

Article Title: Eukaryotic Elongation Factor 1A Interacts with Sphingosine Kinase and Directly Enhances Its Catalytic Activity *
Article Snippet: These beads were then either used directly in pulldown analysis, or the GST fusion proteins eluted by incubation with cold PBS containing 10 m m GSH for 30 min with constant mixing. .. Sphingosine Kinase Assays —Sphingosine kinase activity was routinely determined using d - erythro -sphingosine (Biomol, Plymouth Meeting, PA) and γ-32 P]ATP (PerkinElmer Life Sciences) as substrates, as described previously ( ).

Activity Assay:

Article Title: Active site–adjacent phosphorylation at Tyr-397 by c-Abl kinase inactivates caspase-9
Article Snippet: .. c-Abl (20 μ m ) was incubated in kinase activity buffer (50 m m Tris-Cl, pH 7.5, 20 m m MgCl2 , 0.1 m m EDTA, 0.5 m m EGTA, 5 m m β-glycerophosphate, 1 m m Na3 VO4 ) and allowed to autoactivate in the presence of 250 μ m ATP with [γ-32 P]ATP (10 μCi/μl; PerkinElmer Life Sciences) for 2 h at 30 °C. .. Casp-9 (50 μ m ) was incubated with 1 μ m autoactivated c-Abl in kinase activity buffer with 1 m m ATP containing [γ-32 P]ATP for 4 h at 30 °C.

Article Title: Pathways Leading from BarA/SirA to Motility and Virulence Gene Expression in Salmonella
Article Snippet: .. Phosphorylation and transphosphorylation reactions were carried out by using phosphorylation buffer ( ) with 40 μM [γ-32 P]ATP at room temperature (specific activity, 3,000 Ci/mmol; Perkin-Elmer Life Sciences, Wellesley, Mass.). ..

Article Title: Metabolism of circulating ADP in the bloodstream is mediated via integrated actions of soluble adenylate kinase-1 and NTPDase1/CD39 activities
Article Snippet: Soluble purinergic activities were determined at 37°C in a final volume of 80 μl RPMI 1640 medium supplemented with 25 mM HEPES (pH 7.35) and containing 4 mM β-glycerophosphate and 4 μl murine serum in the following ways: for ADPase assays, serum was pretreated for 20 min with 80 μM diadenosine pentaphosphate (Ap5 A; Sigma, St. Louis, MO, USA), followed by 60 min incubation with 100 μM [2,8-3 H]ADP (Perkin Elmer, Boston, MA, USA); 5′-nucleotidase activity was determined after 60 min incubation of serum with 300 μM [2-3 H]AMP (Quotient Bioresearch; GE Healthcare, Rushden, UK); AK and NDP kinase were assayed by incubating the serum with 400 μM [3 H]AMP (for 45 min) or [3 H]ADP (for 15 min) as respective phosphorus acceptors in the presence of 750 μM γ-phosphate-donating ATP; adenosine deaminase was measured by incubating the serum for 60 min with 300 μM [2-3 H]adenosine (Amersham, Little Chalfont, UK). .. Autoradiographic analysis was also performed by incubating 4 μl murine serum for 30 min in 60 μl RPMI 1640 containing 4 mM β-glycerophosphate, 50 μM [γ-32 P]ATP (Perkin Elmer) and various unlabeled nucleotides, followed by TLC separation of mixture aliquots on Polygram CEL-300 PEI sheets (Macherey-Nagel) with 0.75 M KH2 PO4 (pH 3.5) as solvent.

Article Title: Eukaryotic Elongation Factor 1A Interacts with Sphingosine Kinase and Directly Enhances Its Catalytic Activity *
Article Snippet: .. Sphingosine Kinase Assays —Sphingosine kinase activity was routinely determined using d - erythro -sphingosine (Biomol, Plymouth Meeting, PA) and γ-32 P]ATP (PerkinElmer Life Sciences) as substrates, as described previously ( ). .. A unit (U) of SK activity is defined as the amount of enzyme required to produce 1 pmol of S1P/min.

In Silico:

Article Title: Extracellular Signal-Regulated Kinase Promotes Rho-Dependent Focal Adhesion Formation by Suppressing p190A RhoGAP ▿
Article Snippet: After phosphatase treatment, immunoprecipitates were washed in kinase buffer (20 mM Tris, pH 7.8, 10 mM MgCl2 ,) and suspended in 50 μl of kinase buffer supplemented with 1 mM DTT, 10 μM ATP, 10 μCi [γ-32 P]ATP (EazyTide; Perkin Elmer) and 0 to 100 ng of recombinant ERK1 (MBL International Corporation). .. In silico trypsin digestion was performed with PeptideCutter (ca.expasy.org/tools/peptidecutter).

Mass Spectrometry:

Article Title: Metabolism of circulating ADP in the bloodstream is mediated via integrated actions of soluble adenylate kinase-1 and NTPDase1/CD39 activities
Article Snippet: 3 H-labeled nucleotides and nucleosides were separated by TLC and then either quantified by scintillation β-counting ( ) or exposed to Kodak BioMax MS films (Eastman Kodak, Rochester, NY, USA) for 2 wk at −70°C and developed by autoradiography. .. Autoradiographic analysis was also performed by incubating 4 μl murine serum for 30 min in 60 μl RPMI 1640 containing 4 mM β-glycerophosphate, 50 μM [γ-32 P]ATP (Perkin Elmer) and various unlabeled nucleotides, followed by TLC separation of mixture aliquots on Polygram CEL-300 PEI sheets (Macherey-Nagel) with 0.75 M KH2 PO4 (pH 3.5) as solvent.

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).

Article Title: Transcriptional Corepressors HIPK1 and HIPK2 Control Angiogenesis Via TGF-?-TAK1-Dependent Mechanism
Article Snippet: Immune complexes were washed with kinase buffer (25 mM Tris-HCl, pH 8.0, 10 mM MgCl2 ), and then incubated with 1 mM ATP and 5 µCi of γ-32 P-ATP (Perkin Elmer) for 3 h at room temperature. .. Phosphorylation of HIPK2 on Y361 was confirmed by HIPK2-P-Y361 specific antibody (Thermo Scientific, Cat No. PA5-13045, 1∶500 dilution) in Western blots using HEK293T cell lysates.

Kinase Assay:

Article Title: Transcriptional Corepressors HIPK1 and HIPK2 Control Angiogenesis Via TGF-?-TAK1-Dependent Mechanism
Article Snippet: Paragraph title: Co-IP and In Vitro Kinase Assay ... Immune complexes were washed with kinase buffer (25 mM Tris-HCl, pH 8.0, 10 mM MgCl2 ), and then incubated with 1 mM ATP and 5 µCi of γ-32 P-ATP (Perkin Elmer) for 3 h at room temperature.

Transfection:

Article Title: Transcriptional Corepressors HIPK1 and HIPK2 Control Angiogenesis Via TGF-?-TAK1-Dependent Mechanism
Article Snippet: For in vitro kinase assays, cells were treated with DMSO or 10 ng/ml TGF-β 24 h after transfection, and then whole-cell lysates were collected in lysis buffer. .. Immune complexes were washed with kinase buffer (25 mM Tris-HCl, pH 8.0, 10 mM MgCl2 ), and then incubated with 1 mM ATP and 5 µCi of γ-32 P-ATP (Perkin Elmer) for 3 h at room temperature.

Chromatography:

Article Title: Unencumbered Pol β lyase activity in nucleosome core particles
Article Snippet: Oligomers were either 5′-end-labeled with γ-32 P-ATP (Perkin Elmer) using T4 polynucleotide kinase (Invitrogen) following the manufacturer's specifications or cordycepin and terminal deoxynucleotidyl transferase (Thermo Fisher). .. Excess unincorporated radiolabel (γ-32 P-ATP or cordycepin) was removed using Bio-spin P-30 or P-6 chromatography columns (Biorad).

Activation Assay:

Article Title: DNA-PK Target Identification Reveals Novel Links between DNA Repair Signaling and Cytoskeletal Regulation
Article Snippet: In vitro phosphorylation and autoradiography Vimentin protofilaments and filament complexes were reconstituted from purified vimentin (ab73843-10, Abcam, Cambridge, MA, USA) by stepwise drop dialysis (with “V” Series Membranes (0.025 µm pores, Millipore, Billerica, MA, USA)) of a 1 µg/µl vimentin solution in 9.5 M urea to 4 M urea (4 M urea, 30 mM Tris-HCl pH 7.4, 10 mM ammonium chloride, 2 mM EDTA, 2 mM DTT) then to physiological salt concentration (50 mM NaCl, 2 mM DTT, 10 mM Tris-HCl, pH 7.4) and finally to a DNA-PK activation buffer (20 mM KCl, 0.2 mM DTT, 10 mM HEPES/KOH pH 7.5, 2 mM MgCl2 , 0.04 mM EGTA, 0.02 mM EDTA/KOH pH 7.5). .. We incubated 0–3.5 µg vimentin or 0–10 µg microtubules with 200 U purified DNA-PK (Promega, Lyons, France), 0.1 mM ATP, 0.001 mM (γ-32 P)ATP (10 mCi/ml, Perkin Elmer, Courtaboeuf, France), 10 µg/ml bovine serum albumin (BSA) and 0.1 µg/µl Dbait32Hc or 0.03 µg/µl thymus DNA (Promega) in 20 mM KCl, 0.2 mM DTT, 10 mM HEPES/KOH pH 7.5, 2 mM MgCl2 , 0.04 mM EGTA, 0.02 mM EDTA/KOH pH 7.5.

Protease Inhibitor:

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
Article Snippet: .. The immunoprecipitated samples were dephosphorylated by calf intestinal phosphatase (final concentration of 0.038 U/µL, cat. no. 10713023001, Roche Diagnostics) for 10 minutes and washed 3 times with Buffer C (50 mM Tris HCl, pH 7,4, 10 mM MgCl2 , 0.5% NP-40, 1x protease inhibitor cocktail [cat. no. P8340, Sigma-Aldrich]), followed by labeling with γ-[32 P]-ATP (2.1 µCi, cat. no. NEG502H, Perkin Elmer, Maanstraat, Germany) by polynucleotide kinase phosphorylation (0.5 U/µL, cat. no. M0201L, New England Biolabs Inc., Ipswich, MA) and further washed 3 times with Buffer C. Samples were resolved on 6% denaturing polyacrylamide gels. .. Unsaturated images from the Typhoon 9410 phosphoimager (GE Healthcare Life Sciences) were quantified using the histogram function in Adobe Photoshop (San Jose, CA).

Article Title: Transcriptional Corepressors HIPK1 and HIPK2 Control Angiogenesis Via TGF-?-TAK1-Dependent Mechanism
Article Snippet: Co-IP and In Vitro Kinase Assay Whole-cell lysates were collected from HEK293T cells 24 h after transfection in lysis buffer containing 50 mM HEPES (pH 7.4), 50 mM NaCl, 0.1% Tween 20, 20% glycerol, and 1× protease inhibitor cocktail (Roche Molecular Systems) with brief sonication. .. Immune complexes were washed with kinase buffer (25 mM Tris-HCl, pH 8.0, 10 mM MgCl2 ), and then incubated with 1 mM ATP and 5 µCi of γ-32 P-ATP (Perkin Elmer) for 3 h at room temperature.

Generated:

Article Title: Unencumbered Pol β lyase activity in nucleosome core particles
Article Snippet: Preparation of DNA substrates containing single-nucleotide gaps To generate each of the substrates containing the 147 bp Widom 601 DNA positioning sequence, the damaged strand contained phosphorylated deoxyuridine at the 5′ end of a DNA nick so that a 5′-deoxyribose phosphate (dRP) lyase substrate could be generated upon treatment with Escherichia coli UDG ( ). .. Oligomers were either 5′-end-labeled with γ-32 P-ATP (Perkin Elmer) using T4 polynucleotide kinase (Invitrogen) following the manufacturer's specifications or cordycepin and terminal deoxynucleotidyl transferase (Thermo Fisher).

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).

Imaging:

Article Title: Preformed Soluble Chemoreceptor Trimers That Mimic Cellular Assembly States and Activate CheA Autophosphorylation
Article Snippet: After incubation, 2 μL of 2.3 mM cold ATP and 3–8 μL of a [γ-32 P]ATP (3000 Ci/mmol, 10 mCi/mL, PerkinElmer) solution were added to the sample to produce a total volume of 25 μL. .. The dry gel was placed in an imaging cassette for at least 24 h and then imaged with a Storm phosphorimager (GE Healthcare).

Sequencing:

Article Title: Unencumbered Pol β lyase activity in nucleosome core particles
Article Snippet: Preparation of DNA substrates containing single-nucleotide gaps To generate each of the substrates containing the 147 bp Widom 601 DNA positioning sequence, the damaged strand contained phosphorylated deoxyuridine at the 5′ end of a DNA nick so that a 5′-deoxyribose phosphate (dRP) lyase substrate could be generated upon treatment with Escherichia coli UDG ( ). .. Oligomers were either 5′-end-labeled with γ-32 P-ATP (Perkin Elmer) using T4 polynucleotide kinase (Invitrogen) following the manufacturer's specifications or cordycepin and terminal deoxynucleotidyl transferase (Thermo Fisher).

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
Article Snippet: DNA kinase assays PNKP (10 pmol) was premixed with 40 pmol of full-length XRCC1-His, His-XRCC1FFF , His-XRCC1161–406 , or His-XRCC1161–406 R335A/K369A at 37 °C for 5 min and then the mixtures were added to 20-μl (total volume) reactions containing kinase buffer (80 mm succinic acid, pH 5.5, 10 mm MgCl2 , and 1 mm dithiothreitol), 0.2 nmol of 24-mer 5′-DNA kinase substrate (Integrated DNA Technologies; the single- and double-stranded DNA substrates used in this study have been described previously ( )) and 3.3 pmol of [γ-32 P]ATP (PerkinElmer Life Sciences) and incubated for 2 min at 37 °C. .. 4-μl aliquots were mixed with 2 μl of 3× sequencing gel loading dye (Fisher), boiled for 10 min, and fractionated on a 12% polyacrylamide, 7 m urea sequencing gel at 200 V. Gels were imaged on a Typhoon 9400 variable mode imager (GE Healthcare, Bucks, UK) and quantified using ImageQuant 5.2 software (GE Healthcare).

Sonication:

Article Title: Transcriptional Corepressors HIPK1 and HIPK2 Control Angiogenesis Via TGF-?-TAK1-Dependent Mechanism
Article Snippet: Co-IP and In Vitro Kinase Assay Whole-cell lysates were collected from HEK293T cells 24 h after transfection in lysis buffer containing 50 mM HEPES (pH 7.4), 50 mM NaCl, 0.1% Tween 20, 20% glycerol, and 1× protease inhibitor cocktail (Roche Molecular Systems) with brief sonication. .. Immune complexes were washed with kinase buffer (25 mM Tris-HCl, pH 8.0, 10 mM MgCl2 ), and then incubated with 1 mM ATP and 5 µCi of γ-32 P-ATP (Perkin Elmer) for 3 h at room temperature.

Recombinant:

Article Title: Extracellular Signal-Regulated Kinase Promotes Rho-Dependent Focal Adhesion Formation by Suppressing p190A RhoGAP ▿
Article Snippet: .. After phosphatase treatment, immunoprecipitates were washed in kinase buffer (20 mM Tris, pH 7.8, 10 mM MgCl2 ,) and suspended in 50 μl of kinase buffer supplemented with 1 mM DTT, 10 μM ATP, 10 μCi [γ-32 P]ATP (EazyTide; Perkin Elmer) and 0 to 100 ng of recombinant ERK1 (MBL International Corporation). .. After 30 min of incubation at 30°C, kinase reactions were stopped by adding 2× SDS-PAGE sample buffer and analyzed by PhosphorImager or exposure to X-ray film.

Cleavage Assay:

Article Title: Extent to which hairpin opening by the Artemis:DNA-PKcs complex can contribute to junctional diversity in V(D)J recombination
Article Snippet: .. In vitro nuclease assay For the 3′ overhang cleavage assay, the longer oligonucleotide was labeled with T4 polynucleotide kinase (PNK) and [γ-32 P] ATP (3000 Ci/mmol, PerkinElmer), and then annealed to the partner unlabeled oligonucleotide at equal molar ratios. ..

Nucleic Acid Electrophoresis:

Article Title: Preformed Soluble Chemoreceptor Trimers That Mimic Cellular Assembly States and Activate CheA Autophosphorylation
Article Snippet: After incubation, 2 μL of 2.3 mM cold ATP and 3–8 μL of a [γ-32 P]ATP (3000 Ci/mmol, 10 mCi/mL, PerkinElmer) solution were added to the sample to produce a total volume of 25 μL. .. After [γ-32 P]ATP exposure times from 10 s to 12 min (up to 40 min for TarFO short), the sample was quenched with 25 μL of 3× SDS with 50 mM EDTA (pH 8.0) and then subjected to gel electrophoresis on a 4–20% gradient Tris-glycine gel.

Labeling:

Article Title: Unencumbered Pol β lyase activity in nucleosome core particles
Article Snippet: Oligomers were either 5′-end-labeled with γ-32 P-ATP (Perkin Elmer) using T4 polynucleotide kinase (Invitrogen) following the manufacturer's specifications or cordycepin and terminal deoxynucleotidyl transferase (Thermo Fisher). .. After labeling the strand of interest, the dsDNA was generated by annealing two oligos to a complementary strand, in a 1:1:1 molar ratio, by heating to 95°C for 10 min and slow cooling in annealing buffer containing 10 mM Tris–HCl, pH 8, and 50 mM NaCl.

Article Title: Extracellular Signal-Regulated Kinase Promotes Rho-Dependent Focal Adhesion Formation by Suppressing p190A RhoGAP ▿
Article Snippet: Paragraph title: Metabolic and in vitro labeling. ... After phosphatase treatment, immunoprecipitates were washed in kinase buffer (20 mM Tris, pH 7.8, 10 mM MgCl2 ,) and suspended in 50 μl of kinase buffer supplemented with 1 mM DTT, 10 μM ATP, 10 μCi [γ-32 P]ATP (EazyTide; Perkin Elmer) and 0 to 100 ng of recombinant ERK1 (MBL International Corporation).

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: Extent to which hairpin opening by the Artemis:DNA-PKcs complex can contribute to junctional diversity in V(D)J recombination
Article Snippet: .. In vitro nuclease assay For the 3′ overhang cleavage assay, the longer oligonucleotide was labeled with T4 polynucleotide kinase (PNK) and [γ-32 P] ATP (3000 Ci/mmol, PerkinElmer), and then annealed to the partner unlabeled oligonucleotide at equal molar ratios. ..

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
Article Snippet: .. The immunoprecipitated samples were dephosphorylated by calf intestinal phosphatase (final concentration of 0.038 U/µL, cat. no. 10713023001, Roche Diagnostics) for 10 minutes and washed 3 times with Buffer C (50 mM Tris HCl, pH 7,4, 10 mM MgCl2 , 0.5% NP-40, 1x protease inhibitor cocktail [cat. no. P8340, Sigma-Aldrich]), followed by labeling with γ-[32 P]-ATP (2.1 µCi, cat. no. NEG502H, Perkin Elmer, Maanstraat, Germany) by polynucleotide kinase phosphorylation (0.5 U/µL, cat. no. M0201L, New England Biolabs Inc., Ipswich, MA) and further washed 3 times with Buffer C. Samples were resolved on 6% denaturing polyacrylamide gels. .. Unsaturated images from the Typhoon 9410 phosphoimager (GE Healthcare Life Sciences) were quantified using the histogram function in Adobe Photoshop (San Jose, CA).

Purification:

Article Title: DNA-PK Target Identification Reveals Novel Links between DNA Repair Signaling and Cytoskeletal Regulation
Article Snippet: .. We incubated 0–3.5 µg vimentin or 0–10 µg microtubules with 200 U purified DNA-PK (Promega, Lyons, France), 0.1 mM ATP, 0.001 mM (γ-32 P)ATP (10 mCi/ml, Perkin Elmer, Courtaboeuf, France), 10 µg/ml bovine serum albumin (BSA) and 0.1 µg/µl Dbait32Hc or 0.03 µg/µl thymus DNA (Promega) in 20 mM KCl, 0.2 mM DTT, 10 mM HEPES/KOH pH 7.5, 2 mM MgCl2 , 0.04 mM EGTA, 0.02 mM EDTA/KOH pH 7.5. .. The mixture was incubated for 30 min at 30°C and the reaction was stopped by adding 2×SDS sample buffer to give a final concentration of 50 mM Tris-HCl (pH 6.8), 1% β-mercaptoethanol, 2% SDS, 0.1% bromophenol blue and 10% glycerol.

Nuclease Assay:

Article Title: Extent to which hairpin opening by the Artemis:DNA-PKcs complex can contribute to junctional diversity in V(D)J recombination
Article Snippet: .. In vitro nuclease assay For the 3′ overhang cleavage assay, the longer oligonucleotide was labeled with T4 polynucleotide kinase (PNK) and [γ-32 P] ATP (3000 Ci/mmol, PerkinElmer), and then annealed to the partner unlabeled oligonucleotide at equal molar ratios. ..

Concentration Assay:

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
Article Snippet: .. The immunoprecipitated samples were dephosphorylated by calf intestinal phosphatase (final concentration of 0.038 U/µL, cat. no. 10713023001, Roche Diagnostics) for 10 minutes and washed 3 times with Buffer C (50 mM Tris HCl, pH 7,4, 10 mM MgCl2 , 0.5% NP-40, 1x protease inhibitor cocktail [cat. no. P8340, Sigma-Aldrich]), followed by labeling with γ-[32 P]-ATP (2.1 µCi, cat. no. NEG502H, Perkin Elmer, Maanstraat, Germany) by polynucleotide kinase phosphorylation (0.5 U/µL, cat. no. M0201L, New England Biolabs Inc., Ipswich, MA) and further washed 3 times with Buffer C. Samples were resolved on 6% denaturing polyacrylamide gels. .. Unsaturated images from the Typhoon 9410 phosphoimager (GE Healthcare Life Sciences) were quantified using the histogram function in Adobe Photoshop (San Jose, CA).

Article Title: DNA-PK Target Identification Reveals Novel Links between DNA Repair Signaling and Cytoskeletal Regulation
Article Snippet: In vitro phosphorylation and autoradiography Vimentin protofilaments and filament complexes were reconstituted from purified vimentin (ab73843-10, Abcam, Cambridge, MA, USA) by stepwise drop dialysis (with “V” Series Membranes (0.025 µm pores, Millipore, Billerica, MA, USA)) of a 1 µg/µl vimentin solution in 9.5 M urea to 4 M urea (4 M urea, 30 mM Tris-HCl pH 7.4, 10 mM ammonium chloride, 2 mM EDTA, 2 mM DTT) then to physiological salt concentration (50 mM NaCl, 2 mM DTT, 10 mM Tris-HCl, pH 7.4) and finally to a DNA-PK activation buffer (20 mM KCl, 0.2 mM DTT, 10 mM HEPES/KOH pH 7.5, 2 mM MgCl2 , 0.04 mM EGTA, 0.02 mM EDTA/KOH pH 7.5). .. We incubated 0–3.5 µg vimentin or 0–10 µg microtubules with 200 U purified DNA-PK (Promega, Lyons, France), 0.1 mM ATP, 0.001 mM (γ-32 P)ATP (10 mCi/ml, Perkin Elmer, Courtaboeuf, France), 10 µg/ml bovine serum albumin (BSA) and 0.1 µg/µl Dbait32Hc or 0.03 µg/µl thymus DNA (Promega) in 20 mM KCl, 0.2 mM DTT, 10 mM HEPES/KOH pH 7.5, 2 mM MgCl2 , 0.04 mM EGTA, 0.02 mM EDTA/KOH pH 7.5.

SDS Page:

Article Title: Extracellular Signal-Regulated Kinase Promotes Rho-Dependent Focal Adhesion Formation by Suppressing p190A RhoGAP ▿
Article Snippet: After phosphatase treatment, immunoprecipitates were washed in kinase buffer (20 mM Tris, pH 7.8, 10 mM MgCl2 ,) and suspended in 50 μl of kinase buffer supplemented with 1 mM DTT, 10 μM ATP, 10 μCi [γ-32 P]ATP (EazyTide; Perkin Elmer) and 0 to 100 ng of recombinant ERK1 (MBL International Corporation). .. After 30 min of incubation at 30°C, kinase reactions were stopped by adding 2× SDS-PAGE sample buffer and analyzed by PhosphorImager or exposure to X-ray film.

Article Title: Transcriptional Corepressors HIPK1 and HIPK2 Control Angiogenesis Via TGF-?-TAK1-Dependent Mechanism
Article Snippet: Immune complexes were washed in buffers containing 50 mM HEPES (pH 7.4), 300 mM NaCl, 0.2 mM EDTA, and 1% NP-40 and analyzed on SDS/PAGE. .. Immune complexes were washed with kinase buffer (25 mM Tris-HCl, pH 8.0, 10 mM MgCl2 ), and then incubated with 1 mM ATP and 5 µCi of γ-32 P-ATP (Perkin Elmer) for 3 h at room temperature.

Article Title: DNA-PK Target Identification Reveals Novel Links between DNA Repair Signaling and Cytoskeletal Regulation
Article Snippet: We incubated 0–3.5 µg vimentin or 0–10 µg microtubules with 200 U purified DNA-PK (Promega, Lyons, France), 0.1 mM ATP, 0.001 mM (γ-32 P)ATP (10 mCi/ml, Perkin Elmer, Courtaboeuf, France), 10 µg/ml bovine serum albumin (BSA) and 0.1 µg/µl Dbait32Hc or 0.03 µg/µl thymus DNA (Promega) in 20 mM KCl, 0.2 mM DTT, 10 mM HEPES/KOH pH 7.5, 2 mM MgCl2 , 0.04 mM EGTA, 0.02 mM EDTA/KOH pH 7.5. .. The denatured protein was separated by SDS-PAGE, in a 12% acrylamide/bisacrylamide (35.5/1) gel.

Article Title: Eukaryotic Elongation Factor 1A Interacts with Sphingosine Kinase and Directly Enhances Its Catalytic Activity *
Article Snippet: Protein attached to the GSH-Sepharose was quantitated with Coomassie Brilliant Blue staining following SDS-PAGE using bovine serum albumin as standard. .. Sphingosine Kinase Assays —Sphingosine kinase activity was routinely determined using d - erythro -sphingosine (Biomol, Plymouth Meeting, PA) and γ-32 P]ATP (PerkinElmer Life Sciences) as substrates, as described previously ( ).

Software:

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
Article Snippet: DNA kinase assays PNKP (10 pmol) was premixed with 40 pmol of full-length XRCC1-His, His-XRCC1FFF , His-XRCC1161–406 , or His-XRCC1161–406 R335A/K369A at 37 °C for 5 min and then the mixtures were added to 20-μl (total volume) reactions containing kinase buffer (80 mm succinic acid, pH 5.5, 10 mm MgCl2 , and 1 mm dithiothreitol), 0.2 nmol of 24-mer 5′-DNA kinase substrate (Integrated DNA Technologies; the single- and double-stranded DNA substrates used in this study have been described previously ( )) and 3.3 pmol of [γ-32 P]ATP (PerkinElmer Life Sciences) and incubated for 2 min at 37 °C. .. 4-μl aliquots were mixed with 2 μl of 3× sequencing gel loading dye (Fisher), boiled for 10 min, and fractionated on a 12% polyacrylamide, 7 m urea sequencing gel at 200 V. Gels were imaged on a Typhoon 9400 variable mode imager (GE Healthcare, Bucks, UK) and quantified using ImageQuant 5.2 software (GE Healthcare).

Article Title: DNA-PK Target Identification Reveals Novel Links between DNA Repair Signaling and Cytoskeletal Regulation
Article Snippet: We incubated 0–3.5 µg vimentin or 0–10 µg microtubules with 200 U purified DNA-PK (Promega, Lyons, France), 0.1 mM ATP, 0.001 mM (γ-32 P)ATP (10 mCi/ml, Perkin Elmer, Courtaboeuf, France), 10 µg/ml bovine serum albumin (BSA) and 0.1 µg/µl Dbait32Hc or 0.03 µg/µl thymus DNA (Promega) in 20 mM KCl, 0.2 mM DTT, 10 mM HEPES/KOH pH 7.5, 2 mM MgCl2 , 0.04 mM EGTA, 0.02 mM EDTA/KOH pH 7.5. .. The gel was dried and its storage Phosphor autoradiograph was scanned with a Storm 820 scanner (GE HealthCare) and analyzed with ImageQuant (GE HealthCare) software.

Co-Immunoprecipitation Assay:

Article Title: Transcriptional Corepressors HIPK1 and HIPK2 Control Angiogenesis Via TGF-?-TAK1-Dependent Mechanism
Article Snippet: Paragraph title: Co-IP and In Vitro Kinase Assay ... Immune complexes were washed with kinase buffer (25 mM Tris-HCl, pH 8.0, 10 mM MgCl2 ), and then incubated with 1 mM ATP and 5 µCi of γ-32 P-ATP (Perkin Elmer) for 3 h at room temperature.

In Vitro:

Article Title: Extracellular Signal-Regulated Kinase Promotes Rho-Dependent Focal Adhesion Formation by Suppressing p190A RhoGAP ▿
Article Snippet: Paragraph title: Metabolic and in vitro labeling. ... After phosphatase treatment, immunoprecipitates were washed in kinase buffer (20 mM Tris, pH 7.8, 10 mM MgCl2 ,) and suspended in 50 μl of kinase buffer supplemented with 1 mM DTT, 10 μM ATP, 10 μCi [γ-32 P]ATP (EazyTide; Perkin Elmer) and 0 to 100 ng of recombinant ERK1 (MBL International Corporation).

Article Title: Extent to which hairpin opening by the Artemis:DNA-PKcs complex can contribute to junctional diversity in V(D)J recombination
Article Snippet: .. In vitro nuclease assay For the 3′ overhang cleavage assay, the longer oligonucleotide was labeled with T4 polynucleotide kinase (PNK) and [γ-32 P] ATP (3000 Ci/mmol, PerkinElmer), and then annealed to the partner unlabeled oligonucleotide at equal molar ratios. ..

Article Title: Transcriptional Corepressors HIPK1 and HIPK2 Control Angiogenesis Via TGF-?-TAK1-Dependent Mechanism
Article Snippet: Paragraph title: Co-IP and In Vitro Kinase Assay ... Immune complexes were washed with kinase buffer (25 mM Tris-HCl, pH 8.0, 10 mM MgCl2 ), and then incubated with 1 mM ATP and 5 µCi of γ-32 P-ATP (Perkin Elmer) for 3 h at room temperature.

Article Title: DNA-PK Target Identification Reveals Novel Links between DNA Repair Signaling and Cytoskeletal Regulation
Article Snippet: Paragraph title: In vitro phosphorylation and autoradiography ... We incubated 0–3.5 µg vimentin or 0–10 µg microtubules with 200 U purified DNA-PK (Promega, Lyons, France), 0.1 mM ATP, 0.001 mM (γ-32 P)ATP (10 mCi/ml, Perkin Elmer, Courtaboeuf, France), 10 µg/ml bovine serum albumin (BSA) and 0.1 µg/µl Dbait32Hc or 0.03 µg/µl thymus DNA (Promega) in 20 mM KCl, 0.2 mM DTT, 10 mM HEPES/KOH pH 7.5, 2 mM MgCl2 , 0.04 mM EGTA, 0.02 mM EDTA/KOH pH 7.5.

Article Title: Eukaryotic Elongation Factor 1A Interacts with Sphingosine Kinase and Directly Enhances Its Catalytic Activity *
Article Snippet: Sphingosine Kinase Assays —Sphingosine kinase activity was routinely determined using d - erythro -sphingosine (Biomol, Plymouth Meeting, PA) and γ-32 P]ATP (PerkinElmer Life Sciences) as substrates, as described previously ( ). .. In Vitro Phosphorylation of SK1 and eEF1A — In vitro phosphorylation of GST-SK1 was performed while the protein remained bound to the GSH-Sepharose beads by incubating these beads (2 μg of GST-SK1) with 60 units of ERK2 (Calbiochem) and 1 m m ATP in ERK assay buffer (9 m m MOPS, 11 m m β-glycerophosphate, 2.2 m m EGTA, and 0.4 m m sodium orthovanadate) for 60 min at 30 °C.

Produced:

Article Title: Eukaryotic Elongation Factor 1A Interacts with Sphingosine Kinase and Directly Enhances Its Catalytic Activity *
Article Snippet: GST-SK1 fusion protein were produced as previously described ( ). .. Sphingosine Kinase Assays —Sphingosine kinase activity was routinely determined using d - erythro -sphingosine (Biomol, Plymouth Meeting, PA) and γ-32 P]ATP (PerkinElmer Life Sciences) as substrates, as described previously ( ).

Immunoprecipitation:

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
Article Snippet: .. The immunoprecipitated samples were dephosphorylated by calf intestinal phosphatase (final concentration of 0.038 U/µL, cat. no. 10713023001, Roche Diagnostics) for 10 minutes and washed 3 times with Buffer C (50 mM Tris HCl, pH 7,4, 10 mM MgCl2 , 0.5% NP-40, 1x protease inhibitor cocktail [cat. no. P8340, Sigma-Aldrich]), followed by labeling with γ-[32 P]-ATP (2.1 µCi, cat. no. NEG502H, Perkin Elmer, Maanstraat, Germany) by polynucleotide kinase phosphorylation (0.5 U/µL, cat. no. M0201L, New England Biolabs Inc., Ipswich, MA) and further washed 3 times with Buffer C. Samples were resolved on 6% denaturing polyacrylamide gels. .. Unsaturated images from the Typhoon 9410 phosphoimager (GE Healthcare Life Sciences) were quantified using the histogram function in Adobe Photoshop (San Jose, CA).

Thin Layer Chromatography:

Article Title: Metabolism of circulating ADP in the bloodstream is mediated via integrated actions of soluble adenylate kinase-1 and NTPDase1/CD39 activities
Article Snippet: .. Autoradiographic analysis was also performed by incubating 4 μl murine serum for 30 min in 60 μl RPMI 1640 containing 4 mM β-glycerophosphate, 50 μM [γ-32 P]ATP (Perkin Elmer) and various unlabeled nucleotides, followed by TLC separation of mixture aliquots on Polygram CEL-300 PEI sheets (Macherey-Nagel) with 0.75 M KH2 PO4 (pH 3.5) as solvent. .. In some competitive assays, the samples were pretreated for 20 min with various concentrations of specific AK inhibitor Ap5 A , as well as NTPDase1 inhibitors sodium polyoxotungstate-1 (POM-1; ref. ) and ARL-67156 , and nonselective antagonist of P2 receptors pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS; ref. ) (all from Tocris Bioscience, Bristol, UK), prior to addition of nucleotide substrates.

Lysis:

Article Title: Transcriptional Corepressors HIPK1 and HIPK2 Control Angiogenesis Via TGF-?-TAK1-Dependent Mechanism
Article Snippet: For in vitro kinase assays, cells were treated with DMSO or 10 ng/ml TGF-β 24 h after transfection, and then whole-cell lysates were collected in lysis buffer. .. Immune complexes were washed with kinase buffer (25 mM Tris-HCl, pH 8.0, 10 mM MgCl2 ), and then incubated with 1 mM ATP and 5 µCi of γ-32 P-ATP (Perkin Elmer) for 3 h at room temperature.

Staining:

Article Title: Preformed Soluble Chemoreceptor Trimers That Mimic Cellular Assembly States and Activate CheA Autophosphorylation
Article Snippet: After incubation, 2 μL of 2.3 mM cold ATP and 3–8 μL of a [γ-32 P]ATP (3000 Ci/mmol, 10 mCi/mL, PerkinElmer) solution were added to the sample to produce a total volume of 25 μL. .. The gel was stained with Coomassie blue (10 min), destained with water, and then dried with a GelAir dryer (Bio-Rad).

Article Title: Eukaryotic Elongation Factor 1A Interacts with Sphingosine Kinase and Directly Enhances Its Catalytic Activity *
Article Snippet: Protein attached to the GSH-Sepharose was quantitated with Coomassie Brilliant Blue staining following SDS-PAGE using bovine serum albumin as standard. .. Sphingosine Kinase Assays —Sphingosine kinase activity was routinely determined using d - erythro -sphingosine (Biomol, Plymouth Meeting, PA) and γ-32 P]ATP (PerkinElmer Life Sciences) as substrates, as described previously ( ).

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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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    99
    PerkinElmer atp
    The phosphoadaptor subunit <t>Cks1</t> provide processivity for the multiphosphorylation of Sic1 by Cln2-Cdk1 and Clb5-Cdk1. (a) Cln2- and Clb5-Cdk1 complexes were incubated with Sic1ΔC and 32 <t>P-ATP.</t> The reactions also included wild-type Cks1 (wt) or a version with a mutated phosphate-binding site ( mut ; see Supplementary Methods ). Phosphorylated substrates were separated using Phos-Tag SDS-PAGE gels. (b) Reactions were performed in the presence of a phosphopeptide competitor (P) based on the sequence surrounding T45 in Sic1. (c) The phosphorylation of a Sic1ΔC version containing a single Cdk site (Sic1ΔC-T5, with other Cdk consensus sites mutated to alanines) was not affected by Cks1 mut or the phosphopeptide. The standard SDS-PAGE was used. (d) Time courses of Sic1ΔC multiphosphorylation were followed by Phos-Tag SDS-PAGE. (e) The quantified data from (d). The intensities of 32 P-labeled proteins were divided by the number of phosphates as indicated to obtain the levels of different phosphoforms. In the experiments presented in Fig. 1 the enzyme concentrations were chosen to obtain roughly equal substrate labeling.
    Atp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    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

    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.

    Journal: Molecular Biology of the Cell

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

    doi: 10.1091/mbc.E10-07-0636

    Figure Lengend 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.

    Article Snippet: In Vitro Kinase Assays pET and pET-Aly2 were incubated for 30 min at 30°C in kinase buffer (50 mM Tris-HCl, pH 7.5, 20 mM MgCl2 , 1 mM DTT, 1 μM unlabeled ATP, aprotinin, and leupeptin) with 75 nM [γ-32 P]ATP (Perkin Elmer-Cetus) with or without Npr1 kinase (purified from Y258 yeast cells).

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

    The phosphoadaptor subunit Cks1 provide processivity for the multiphosphorylation of Sic1 by Cln2-Cdk1 and Clb5-Cdk1. (a) Cln2- and Clb5-Cdk1 complexes were incubated with Sic1ΔC and 32 P-ATP. The reactions also included wild-type Cks1 (wt) or a version with a mutated phosphate-binding site ( mut ; see Supplementary Methods ). Phosphorylated substrates were separated using Phos-Tag SDS-PAGE gels. (b) Reactions were performed in the presence of a phosphopeptide competitor (P) based on the sequence surrounding T45 in Sic1. (c) The phosphorylation of a Sic1ΔC version containing a single Cdk site (Sic1ΔC-T5, with other Cdk consensus sites mutated to alanines) was not affected by Cks1 mut or the phosphopeptide. The standard SDS-PAGE was used. (d) Time courses of Sic1ΔC multiphosphorylation were followed by Phos-Tag SDS-PAGE. (e) The quantified data from (d). The intensities of 32 P-labeled proteins were divided by the number of phosphates as indicated to obtain the levels of different phosphoforms. In the experiments presented in Fig. 1 the enzyme concentrations were chosen to obtain roughly equal substrate labeling.

    Journal: Nature

    Article Title: Cascades of multisite phosphorylation control Sic1 destruction at the onset of S phase

    doi: 10.1038/nature10560

    Figure Lengend Snippet: The phosphoadaptor subunit Cks1 provide processivity for the multiphosphorylation of Sic1 by Cln2-Cdk1 and Clb5-Cdk1. (a) Cln2- and Clb5-Cdk1 complexes were incubated with Sic1ΔC and 32 P-ATP. The reactions also included wild-type Cks1 (wt) or a version with a mutated phosphate-binding site ( mut ; see Supplementary Methods ). Phosphorylated substrates were separated using Phos-Tag SDS-PAGE gels. (b) Reactions were performed in the presence of a phosphopeptide competitor (P) based on the sequence surrounding T45 in Sic1. (c) The phosphorylation of a Sic1ΔC version containing a single Cdk site (Sic1ΔC-T5, with other Cdk consensus sites mutated to alanines) was not affected by Cks1 mut or the phosphopeptide. The standard SDS-PAGE was used. (d) Time courses of Sic1ΔC multiphosphorylation were followed by Phos-Tag SDS-PAGE. (e) The quantified data from (d). The intensities of 32 P-labeled proteins were divided by the number of phosphates as indicated to obtain the levels of different phosphoforms. In the experiments presented in Fig. 1 the enzyme concentrations were chosen to obtain roughly equal substrate labeling.

    Article Snippet: The general composition of the assay mixture was as follows: 50 mM Hepes pH 7.4, 100 mM NaCl, 0.1% NP-40, 20 mM imidazole, 2% glycerol, 2 mM EGTA, 0.2 mg/ml BSA, 500 nM Cks1 and 500 μM ATP (with added γ-32 P-ATP (Perkin Elmer)).

    Techniques: Incubation, Binding Assay, SDS Page, Sequencing, Labeling