Structured Review

PerkinElmer γ 32 p atp
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 <t>P]ATP</t> 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.
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Images

1) 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

2) 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

3) 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

4) 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

5) 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

6) 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

7) 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

8) 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

9) 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

10) 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

11) 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

12) 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

13) 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

14) 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

15) 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

16) 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

17) 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

18) 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

19) 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

20) 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

21) 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

22) 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

23) 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

24) 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

25) 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

26) 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

27) Product Images from "Activity, Abundance, and Localization of Quorum Sensing Receptors in Vibrio harveyi"

Article Title: Activity, Abundance, and Localization of Quorum Sensing Receptors in Vibrio harveyi

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2017.00634

In vitro enzymatic activities of the QS receptors LuxN, Lux(P)Q, and CqsS-F175C. (A) Time-dependent autophosphorylation of LuxN, Lux(P)Q and CqsS-F175C, and subsequent phosphotransfer to the HPt protein LuxU. Membrane vesicles containing one of the three QS receptors (all hybrid histidine kinases) LuxN, LuxQ, or CqsS-F175C, respectively, were mixed with LuxU, and the reaction was started with [γ 32 -P] Mg 2+ ATP. For LuxQ mediated phosphorylation, LuxP had been incorporated into the vesicles. At the indicated time points the phosphorylation reaction was stopped, proteins were separated by SDS-PAGE followed by exposure of the gels to a phosphoscreen. The autoradiograph corresponding to P-LuxU protein size is representative for three independent experiments. (B) Quantitative analysis of the data presented in (A) . Bands corresponding to P-LuxU were quantified using ImageQuant, and relative values were calculated (The amount P-LuxU at 0.5 min of phosphorylation was normalized to 1). (C) Influence of AIs on the kinase activity of the QS receptors. When indicated HAI-1, AI-2, or CAI-1 (from V. cholerae ) was added to the assay mixture (described in A ) in a concentration of 10 μM prior incubation. Ten minutes after incubation the reaction was stopped and P-LuxU was quantified. (D) Dephosphorylation of P-LuxU. A mixture of P-LuxU and LuxN, Lux(P)Q, or CqsS-F175C, respectively, was incubated. At the indicated time points the reactions were stopped, proteins were separated via SDS-PAGE, and the amount of P-LuxU was quantified. Relative values are presented, and the amount of P-LuxU in the absence of the hybrid histidine kinases was set to 1. All quantifications were performed with ImageQuant (GE Healthcare, Munich, Germany) using [γ 32 -P] Mg 2+ ATP as standard. Shown are the mean values and standard deviations of three independent experiments.
Figure Legend Snippet: In vitro enzymatic activities of the QS receptors LuxN, Lux(P)Q, and CqsS-F175C. (A) Time-dependent autophosphorylation of LuxN, Lux(P)Q and CqsS-F175C, and subsequent phosphotransfer to the HPt protein LuxU. Membrane vesicles containing one of the three QS receptors (all hybrid histidine kinases) LuxN, LuxQ, or CqsS-F175C, respectively, were mixed with LuxU, and the reaction was started with [γ 32 -P] Mg 2+ ATP. For LuxQ mediated phosphorylation, LuxP had been incorporated into the vesicles. At the indicated time points the phosphorylation reaction was stopped, proteins were separated by SDS-PAGE followed by exposure of the gels to a phosphoscreen. The autoradiograph corresponding to P-LuxU protein size is representative for three independent experiments. (B) Quantitative analysis of the data presented in (A) . Bands corresponding to P-LuxU were quantified using ImageQuant, and relative values were calculated (The amount P-LuxU at 0.5 min of phosphorylation was normalized to 1). (C) Influence of AIs on the kinase activity of the QS receptors. When indicated HAI-1, AI-2, or CAI-1 (from V. cholerae ) was added to the assay mixture (described in A ) in a concentration of 10 μM prior incubation. Ten minutes after incubation the reaction was stopped and P-LuxU was quantified. (D) Dephosphorylation of P-LuxU. A mixture of P-LuxU and LuxN, Lux(P)Q, or CqsS-F175C, respectively, was incubated. At the indicated time points the reactions were stopped, proteins were separated via SDS-PAGE, and the amount of P-LuxU was quantified. Relative values are presented, and the amount of P-LuxU in the absence of the hybrid histidine kinases was set to 1. All quantifications were performed with ImageQuant (GE Healthcare, Munich, Germany) using [γ 32 -P] Mg 2+ ATP as standard. Shown are the mean values and standard deviations of three independent experiments.

Techniques Used: In Vitro, SDS Page, Autoradiography, Activity Assay, Concentration Assay, Incubation, De-Phosphorylation Assay

28) Product Images from "Transgenic Biosynthesis of Trypanothione Protects Escherichia coli from Radiation-Induced Toxicity"

Article Title: Transgenic Biosynthesis of Trypanothione Protects Escherichia coli from Radiation-Induced Toxicity

Journal: Radiation research

doi: 10.1667/RR2235.1

Trypanothione protects E. coli from radiation-induced reactive oxygen species accumulation and DNA damage. pETA/TS and pETK/TR-transformed E. coli were grown to 0.6 OD in liquid LB cultures and then treated with 0.25 m M IPTG. Panel A: One hour after γ irradiation, bacteria were incubated with 20 m M H 2 DCF-DA for 30 min at 37°C in the dark and the fluorescence of the oxidative sensitive probe was measured. Bacteria treated with 1 μ M H 2 O 2 were used as a pro-oxidant positive control. Fluorescence intensity is shown as the average measurement of duplicate determinations from a representative experiment; error bars represent the standard deviations. Panel B: DNA was extracted from pETA/TS and pETK/TR-transformed E. coli 2 h after γ irradiation and quantified by UV spectrophotometry. Strand breaks in circular bacterial genomic DNA were enzymatically end-labeled with T4 PNK in the presence of [γ- 32 P]ATP. γ- 32 P incorporation into end-labeled DNA was measured using a scintillation counter. Data are averages of duplicate determinations from a representative experiment; error bars are standard deviations.
Figure Legend Snippet: Trypanothione protects E. coli from radiation-induced reactive oxygen species accumulation and DNA damage. pETA/TS and pETK/TR-transformed E. coli were grown to 0.6 OD in liquid LB cultures and then treated with 0.25 m M IPTG. Panel A: One hour after γ irradiation, bacteria were incubated with 20 m M H 2 DCF-DA for 30 min at 37°C in the dark and the fluorescence of the oxidative sensitive probe was measured. Bacteria treated with 1 μ M H 2 O 2 were used as a pro-oxidant positive control. Fluorescence intensity is shown as the average measurement of duplicate determinations from a representative experiment; error bars represent the standard deviations. Panel B: DNA was extracted from pETA/TS and pETK/TR-transformed E. coli 2 h after γ irradiation and quantified by UV spectrophotometry. Strand breaks in circular bacterial genomic DNA were enzymatically end-labeled with T4 PNK in the presence of [γ- 32 P]ATP. γ- 32 P incorporation into end-labeled DNA was measured using a scintillation counter. Data are averages of duplicate determinations from a representative experiment; error bars are standard deviations.

Techniques Used: Transformation Assay, Irradiation, Incubation, Fluorescence, Positive Control, Spectrophotometry, Labeling

29) Product Images from "Identification of autophosphorylation sites in eukaryotic elongation factor-2 kinase"

Article Title: Identification of autophosphorylation sites in eukaryotic elongation factor-2 kinase

Journal: Biochemical Journal

doi: 10.1042/BJ20111530

Recombinant eEF2K undergoes Ca 2+ /CaM-dependent autophosphorylation ( A ) GST–eEF2K was incubated with [γ- 32 P]ATP in the presence or absence of Ca 2+ /CaM as described in the Experimental section. At the indicated times, samples were analysed by SDS/PAGE for measurements of 32 P incorporation. The values are means±S.E.M. ( n =3). ( B ) Autophosphorylation was measured as in ( A ) with the indicated concentrations of wild-type (WT) eEF2K and eEF2K[S78A] mutant.
Figure Legend Snippet: Recombinant eEF2K undergoes Ca 2+ /CaM-dependent autophosphorylation ( A ) GST–eEF2K was incubated with [γ- 32 P]ATP in the presence or absence of Ca 2+ /CaM as described in the Experimental section. At the indicated times, samples were analysed by SDS/PAGE for measurements of 32 P incorporation. The values are means±S.E.M. ( n =3). ( B ) Autophosphorylation was measured as in ( A ) with the indicated concentrations of wild-type (WT) eEF2K and eEF2K[S78A] mutant.

Techniques Used: Recombinant, Chick Chorioallantoic Membrane Assay, Incubation, SDS Page, Mutagenesis

30) Product Images from "Inactivation and Disassembly of the Anaphase-Promoting Complex during Human Cytomegalovirus Infection Is Associated with Degradation of the APC5 and APC4 Subunits and Does Not Require UL97-Mediated Phosphorylation of Cdh1 ▿"

Article Title: Inactivation and Disassembly of the Anaphase-Promoting Complex during Human Cytomegalovirus Infection Is Associated with Degradation of the APC5 and APC4 Subunits and Does Not Require UL97-Mediated Phosphorylation of Cdh1 ▿

Journal: Journal of Virology

doi: 10.1128/JVI.01260-10

UL97 phosphorylates Cdh1 at multiple sites in vitro . (A) In vitro kinase assays using purified Cdh1 and GST-UL97 were performed with [γ- 32 P]ATP in the presence or absence of maribavir (MBV). 32 P incorporation was imaged on a phosphor screen, and
Figure Legend Snippet: UL97 phosphorylates Cdh1 at multiple sites in vitro . (A) In vitro kinase assays using purified Cdh1 and GST-UL97 were performed with [γ- 32 P]ATP in the presence or absence of maribavir (MBV). 32 P incorporation was imaged on a phosphor screen, and

Techniques Used: In Vitro, Purification

31) Product Images from "AMPK antagonizes hepatic glucagon-stimulated cyclic AMP signalling via phosphorylation-induced activation of cyclic nucleotide phosphodiesterase 4B"

Article Title: AMPK antagonizes hepatic glucagon-stimulated cyclic AMP signalling via phosphorylation-induced activation of cyclic nucleotide phosphodiesterase 4B

Journal: Nature Communications

doi: 10.1038/ncomms10856

Phosphorylation-induced activation of mouse liver PDE4B. PDE4B was cloned from mouse hepatocyte cDNA. The recombinant protein was overexpressed in E. coli and purified. PDE protein was phosphorylated for 1 h with purified recombinant activated AMPK and/or purified PKA catalytic subunits and [γ- 32 P] ATP, and analysed by SDS–PAGE followed by Coomassie blue staining and phosphorimaging for quantification ( a , c ). In b , PDE was phosphorylated for 1 h with recombinant activated AMPK and [γ- 32 P]. Phosphorylation sites were identified by LC–MS/MS after trypsin digestion and radioactive peak separation by high-performance liquid chromatography (HPLC). The phosphorylation sites that were identified are underlined in the right hand panel. In d and e , recombinant PDE was phosphorylated as above but with non-radioactive ATP for PDE assay as indicated. In d , separate determinations of V max and K M were made by linear regression of double reciprocal (Lineweaver Burk) plots. In e , the basal PDE activities of the wild-type (WT), S118A, S125A and S304A mutant proteins were 1.97±0.25, 0.14±0.01, 1.59±0.15 and 0.32±0.09 mU per mg of protein, respectively. Values are means±s.e.m. for n =3 ( c – e ) separate experiments. Statistical analysis was by a paired Student's t -test. *Indicates a significant difference ( P
Figure Legend Snippet: Phosphorylation-induced activation of mouse liver PDE4B. PDE4B was cloned from mouse hepatocyte cDNA. The recombinant protein was overexpressed in E. coli and purified. PDE protein was phosphorylated for 1 h with purified recombinant activated AMPK and/or purified PKA catalytic subunits and [γ- 32 P] ATP, and analysed by SDS–PAGE followed by Coomassie blue staining and phosphorimaging for quantification ( a , c ). In b , PDE was phosphorylated for 1 h with recombinant activated AMPK and [γ- 32 P]. Phosphorylation sites were identified by LC–MS/MS after trypsin digestion and radioactive peak separation by high-performance liquid chromatography (HPLC). The phosphorylation sites that were identified are underlined in the right hand panel. In d and e , recombinant PDE was phosphorylated as above but with non-radioactive ATP for PDE assay as indicated. In d , separate determinations of V max and K M were made by linear regression of double reciprocal (Lineweaver Burk) plots. In e , the basal PDE activities of the wild-type (WT), S118A, S125A and S304A mutant proteins were 1.97±0.25, 0.14±0.01, 1.59±0.15 and 0.32±0.09 mU per mg of protein, respectively. Values are means±s.e.m. for n =3 ( c – e ) separate experiments. Statistical analysis was by a paired Student's t -test. *Indicates a significant difference ( P

Techniques Used: Activation Assay, Clone Assay, Recombinant, Purification, SDS Page, Staining, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, High Performance Liquid Chromatography, Mutagenesis

32) Product Images from "CaMKII uses GTP as a phosphate donor for both substrate and autophosphorylation"

Article Title: CaMKII uses GTP as a phosphate donor for both substrate and autophosphorylation

Journal: Biochemical and biophysical research communications

doi: 10.1016/j.bbrc.2009.10.107

Partial proteolysis of Drosophila CaMKII phosphorylated with ATP and GTP produces identical autophosphorylated fragments. CaMKII was autophosphorylated with γ- 32 P ATP or GTP and excised from a 9% gel after identification by autoradiography. Protein
Figure Legend Snippet: Partial proteolysis of Drosophila CaMKII phosphorylated with ATP and GTP produces identical autophosphorylated fragments. CaMKII was autophosphorylated with γ- 32 P ATP or GTP and excised from a 9% gel after identification by autoradiography. Protein

Techniques Used: Autoradiography

33) Product Images from "Histone H3 Threonine 11 Phosphorylation Is Catalyzed Directly by the Meiosis-Specific Kinase Mek1 and Provides a Molecular Readout of Mek1 Activity in Vivo"

Article Title: Histone H3 Threonine 11 Phosphorylation Is Catalyzed Directly by the Meiosis-Specific Kinase Mek1 and Provides a Molecular Readout of Mek1 Activity in Vivo

Journal: Genetics

doi: 10.1534/genetics.117.300359

H3 T11 is a direct target of Mek1 kinase. (A) Persistence of H3 T11ph requires maintenance of Mek1 kinase activity. A meiotic culture of mek1-as , dmc1 Δ (strain SKY3095) was split 4 hr after transfer to sporulation medium. One part was left to continue in meiosis untreated, the other part was treated with 1 μM 1-NA-PP1. WCEs were prepared at the indicated times and assayed for H3 T11ph by western blotting (mAb; EMD Millipore 05-789). ▴ and ▵ indicate 20- and 15-kDa molecular weight markers, respectively. Numbers indicate hours after transfer to sporulation medium. (B) Mek1 kinase assay using radioactive ATP. Affinity-purified GST-Mek1 (250 ng) was incubated in the presence of [γ- 32 P]ATP either alone or with 2 μg recombinant H3 or 5 μg of unphosphorylated or phosphorylated synthetic H3 1–20 peptides as substrates. Reactions were separated by SDS-PAGE and visualized by autoradiography (top), anti-H3 T11ph western blot (middle; polyclonal Active Motif 39151), and Coomassie staining. (C) Mek1 kinase assay by semisynthetic epitope labeling. Kinase reactions were carried out with affinity-purified GST-Mek1 (2 μg) or GST-Mek1-as (0.76 μg) in the presence of ATPγS or 6-Fu-ATPγS with 2 μg recombinant H3. After incubation 30 min at 30°, PNBM was added to alkylate the thiophosphorylated target sites. Reactions were then separated by SDS-PAGE and analyzed by western blotting with anti-thiophosphate ester mAb (top panel; Epitomics 2686-1). Because ATP was also included in all reactions, samples were also analyzed by western blotting with anti-H3 T11ph mAb (EMD Millipore 05-789) to detect the subset of H3 proteins modified on T11 with phosphate instead of thiophosphate. Note that 1-NA-PP1 inhibits ability of GST-Mek1-as1 to use either ATP or Fu-ATPγS to phosphorylate H3 T11 (lane 4). A shorter exposure of lane 2 of the anti-H3 T11ph blot is shown to the right (* indicates an H3 fragment present in the recombinant histone sample). Interstitial lanes were removed from images in (B and C) as indicated by the white lines. WB, western blot.
Figure Legend Snippet: H3 T11 is a direct target of Mek1 kinase. (A) Persistence of H3 T11ph requires maintenance of Mek1 kinase activity. A meiotic culture of mek1-as , dmc1 Δ (strain SKY3095) was split 4 hr after transfer to sporulation medium. One part was left to continue in meiosis untreated, the other part was treated with 1 μM 1-NA-PP1. WCEs were prepared at the indicated times and assayed for H3 T11ph by western blotting (mAb; EMD Millipore 05-789). ▴ and ▵ indicate 20- and 15-kDa molecular weight markers, respectively. Numbers indicate hours after transfer to sporulation medium. (B) Mek1 kinase assay using radioactive ATP. Affinity-purified GST-Mek1 (250 ng) was incubated in the presence of [γ- 32 P]ATP either alone or with 2 μg recombinant H3 or 5 μg of unphosphorylated or phosphorylated synthetic H3 1–20 peptides as substrates. Reactions were separated by SDS-PAGE and visualized by autoradiography (top), anti-H3 T11ph western blot (middle; polyclonal Active Motif 39151), and Coomassie staining. (C) Mek1 kinase assay by semisynthetic epitope labeling. Kinase reactions were carried out with affinity-purified GST-Mek1 (2 μg) or GST-Mek1-as (0.76 μg) in the presence of ATPγS or 6-Fu-ATPγS with 2 μg recombinant H3. After incubation 30 min at 30°, PNBM was added to alkylate the thiophosphorylated target sites. Reactions were then separated by SDS-PAGE and analyzed by western blotting with anti-thiophosphate ester mAb (top panel; Epitomics 2686-1). Because ATP was also included in all reactions, samples were also analyzed by western blotting with anti-H3 T11ph mAb (EMD Millipore 05-789) to detect the subset of H3 proteins modified on T11 with phosphate instead of thiophosphate. Note that 1-NA-PP1 inhibits ability of GST-Mek1-as1 to use either ATP or Fu-ATPγS to phosphorylate H3 T11 (lane 4). A shorter exposure of lane 2 of the anti-H3 T11ph blot is shown to the right (* indicates an H3 fragment present in the recombinant histone sample). Interstitial lanes were removed from images in (B and C) as indicated by the white lines. WB, western blot.

Techniques Used: Activity Assay, Western Blot, Molecular Weight, Kinase Assay, Affinity Purification, Incubation, Recombinant, SDS Page, Autoradiography, Staining, Labeling, Modification

34) Product Images from "Genetic and Biochemical Analysis of PadR-padC Promoter Interactions during the Phenolic Acid Stress Response in Bacillus subtilis 168 ▿ 168 ▿ ¶"

Article Title: Genetic and Biochemical Analysis of PadR-padC Promoter Interactions during the Phenolic Acid Stress Response in Bacillus subtilis 168 ▿ 168 ▿ ¶

Journal: Journal of Bacteriology

doi: 10.1128/JB.00385-11

padC ( yveFG-padC ) promoter DNA protection with PadR. Purified PadR was incubated with a 234-bp single-end-labeled padC promoter DNA probe. The reaction mixtures contained a 5′-[γ- 32 P]ATP-labeled coding strand (BSD1 primer) (A) or noncoding
Figure Legend Snippet: padC ( yveFG-padC ) promoter DNA protection with PadR. Purified PadR was incubated with a 234-bp single-end-labeled padC promoter DNA probe. The reaction mixtures contained a 5′-[γ- 32 P]ATP-labeled coding strand (BSD1 primer) (A) or noncoding

Techniques Used: Purification, Incubation, Labeling

35) Product Images from "The Stress-Sensing TORC2 Complex Activates Yeast AGC-Family Protein Kinase Ypk1 at Multiple Novel Sites"

Article Title: The Stress-Sensing TORC2 Complex Activates Yeast AGC-Family Protein Kinase Ypk1 at Multiple Novel Sites

Journal: Genetics

doi: 10.1534/genetics.117.1124

TORC2 phosphorylates the novel C-terminal sites. (A) WT (BY4741) or otherwise isogenic tor2 ts cells expressing Ypk1 5A -myc (pFR246) were grown at 26° to midexponential phase and then either kept at 26° or shifted to 37° for 2 hr, harvested, lysed, and samples of the resulting extracts resolved by Phos-tag SDS-PAGE and analyzed by immunoblotting with anti-myc mAb 9E10. (B) TOR1-1 avo3 ∆ CT cells (left) expressing Ypk1 5A -myc (pFR246) were grown to midexponential phase, treated for 20 min with either vehicle alone (Tween 20:ethanol, 10:90) or 200 nM rapamycin in the same solvent as indicated, collected, and analyzed as in (A). WT cells (BY4741) expressing Ypk1 5A -myc (pFR246) (right) were treated in the same manner in a separate experiment. (C) Strain yAM135-A ( ypk1 as ypk2 ∆) expressing Ypk1 5A -myc (pFR246) was grown to midexponential phase and then treated with either vehicle alone (DMSO) or 10 µM 3-MB-PP1 for 1 hr, harvested, and then analyzed as in (A). (D) WT (Mock IP) (yKL4) and Avo3-3C-3XFLAG (TORC2 IP) (yNM695) strains were grown in YPD to midexponential phase, harvested, lysed, and TORC2 immunoprecipitated from the resulting extracts using anti-FLAG antibody-coupled agarose resin. Immunoprecipitated proteins were resolved by SDS-PAGE and analyzed by immunoblotting with anti-Avo3 and anti-Tor2 antibodies. (E) Mock and TORC2 preparations, as in (D), were incubated with [γ- 32 P]ATP, either alone or in the presence of purified analog-sensitive Ypk1 as , in either the absence or presence of the TORC2 inhibitor NVP-BEZ235, as indicated. Reaction products were resolved by SDS-PAGE and analyzed by Coomassie staining and, after drying the gel, by autoradiography.
Figure Legend Snippet: TORC2 phosphorylates the novel C-terminal sites. (A) WT (BY4741) or otherwise isogenic tor2 ts cells expressing Ypk1 5A -myc (pFR246) were grown at 26° to midexponential phase and then either kept at 26° or shifted to 37° for 2 hr, harvested, lysed, and samples of the resulting extracts resolved by Phos-tag SDS-PAGE and analyzed by immunoblotting with anti-myc mAb 9E10. (B) TOR1-1 avo3 ∆ CT cells (left) expressing Ypk1 5A -myc (pFR246) were grown to midexponential phase, treated for 20 min with either vehicle alone (Tween 20:ethanol, 10:90) or 200 nM rapamycin in the same solvent as indicated, collected, and analyzed as in (A). WT cells (BY4741) expressing Ypk1 5A -myc (pFR246) (right) were treated in the same manner in a separate experiment. (C) Strain yAM135-A ( ypk1 as ypk2 ∆) expressing Ypk1 5A -myc (pFR246) was grown to midexponential phase and then treated with either vehicle alone (DMSO) or 10 µM 3-MB-PP1 for 1 hr, harvested, and then analyzed as in (A). (D) WT (Mock IP) (yKL4) and Avo3-3C-3XFLAG (TORC2 IP) (yNM695) strains were grown in YPD to midexponential phase, harvested, lysed, and TORC2 immunoprecipitated from the resulting extracts using anti-FLAG antibody-coupled agarose resin. Immunoprecipitated proteins were resolved by SDS-PAGE and analyzed by immunoblotting with anti-Avo3 and anti-Tor2 antibodies. (E) Mock and TORC2 preparations, as in (D), were incubated with [γ- 32 P]ATP, either alone or in the presence of purified analog-sensitive Ypk1 as , in either the absence or presence of the TORC2 inhibitor NVP-BEZ235, as indicated. Reaction products were resolved by SDS-PAGE and analyzed by Coomassie staining and, after drying the gel, by autoradiography.

Techniques Used: Expressing, SDS Page, Immunoprecipitation, Incubation, Purification, Staining, Autoradiography

36) Product Images from "The multifunctional Ca2+/calmodulin-dependent protein kinase II delta (CaMKII?) phosphorylates cardiac titin's spring elements"

Article Title: The multifunctional Ca2+/calmodulin-dependent protein kinase II delta (CaMKII?) phosphorylates cardiac titin's spring elements

Journal: Journal of molecular and cellular cardiology

doi: 10.1016/j.yjmcc.2012.11.012

The N2B element of cardiac titin is phosphorylated by CaMKIIδ A) Domain organization of cardiac titin’s I-band region (N2B isoform) and location of recombinant proteins used in kinase assay: Ig8-15, Ig84-91, and the N2B element (both the human and murine versions were used). B) Upper panel: Coomassie blue (CB) stained gel. Lower panel: corresponding autoradiograph (AR) of recombinant proteins incubated in kinase buffer (containing Ca 2+ /Calmodulin, and [γ- 32 P]ATP) without (−) and with CaMKIIδ (+). C) Conserved Serines that are phosphorylated by CaMKIIδ (numbering based on N2B unique sequence in human).
Figure Legend Snippet: The N2B element of cardiac titin is phosphorylated by CaMKIIδ A) Domain organization of cardiac titin’s I-band region (N2B isoform) and location of recombinant proteins used in kinase assay: Ig8-15, Ig84-91, and the N2B element (both the human and murine versions were used). B) Upper panel: Coomassie blue (CB) stained gel. Lower panel: corresponding autoradiograph (AR) of recombinant proteins incubated in kinase buffer (containing Ca 2+ /Calmodulin, and [γ- 32 P]ATP) without (−) and with CaMKIIδ (+). C) Conserved Serines that are phosphorylated by CaMKIIδ (numbering based on N2B unique sequence in human).

Techniques Used: Recombinant, Kinase Assay, Staining, Autoradiography, Incubation, Sequencing

Titin is phosphorylated by CaMKIIδ A) Mouse skinned LV muscle fibers incubated with CaMKIIδ. Left panel: Coomassie blue (CB) stained 2–7% gradient gel. Right panel: corresponding autoradiograph (AR). Lane 1: skinned fibers in kinase buffer (containing Ca 2+ /Calmodulin, and [γ- 32 P]ATP). Lane 2: skinned fibers in kinase buffer plus CaMKIIδ. B) Effect of pre-incubation of mouse skinned cardiac fibers with protein phosphatase 1 (PP1). Top: lane 1 (Ctrl) skinned fibers incubated with CaMKIIδ. Lane 2 (PP1) skinned fibers pre-treated with PP1 and then incubated with CaMKIIδ. Upper panel: Coomassie blue (CB) stained gel; Lower panel: corresponding autoradiograph (AR). C) Left: pre-incubation with PP1 resulted in a significant increase in CaMKIIδ induced 32 P incorporation on titin. Right: Phosphorylation level of CaMKIIδ accessible sites under control conditions (expressed as percentage of 32 P incorporation on titin in PP1 pretreated and then CaMKIIδ phosphorylated skinned muscle). (N=12, *** p
Figure Legend Snippet: Titin is phosphorylated by CaMKIIδ A) Mouse skinned LV muscle fibers incubated with CaMKIIδ. Left panel: Coomassie blue (CB) stained 2–7% gradient gel. Right panel: corresponding autoradiograph (AR). Lane 1: skinned fibers in kinase buffer (containing Ca 2+ /Calmodulin, and [γ- 32 P]ATP). Lane 2: skinned fibers in kinase buffer plus CaMKIIδ. B) Effect of pre-incubation of mouse skinned cardiac fibers with protein phosphatase 1 (PP1). Top: lane 1 (Ctrl) skinned fibers incubated with CaMKIIδ. Lane 2 (PP1) skinned fibers pre-treated with PP1 and then incubated with CaMKIIδ. Upper panel: Coomassie blue (CB) stained gel; Lower panel: corresponding autoradiograph (AR). C) Left: pre-incubation with PP1 resulted in a significant increase in CaMKIIδ induced 32 P incorporation on titin. Right: Phosphorylation level of CaMKIIδ accessible sites under control conditions (expressed as percentage of 32 P incorporation on titin in PP1 pretreated and then CaMKIIδ phosphorylated skinned muscle). (N=12, *** p

Techniques Used: Incubation, Staining, Autoradiography

Evaluation of PEVK S26 and PEVK S170 as CaMKIIδ phosphorylation sites ]. Results show that both sites are phosphorylated by CaMKIIδ. B) Proteins were incubated in kinase buffer (containing Ca 2+ /Calmodulin, and [γ- 32 P]ATP) without (−) and with CaMKIIδ (+). Proteins were then electrophoresed, Coomassie blue stained (top), and exposed to autoradiographic film (bottom). Top: phosphorylation results in incorporation of 32 P in all proteins, but the level varies and is highest in the WT protein. Bottom: Quantitative analysis of 32 P incorporation (normalized to protein loading) shows that the single mutants incorporate only about half the WT amount. The phosphorylation level in the double mutant is ~75% less than in WT PEVK. See text for details. (N=5, * p
Figure Legend Snippet: Evaluation of PEVK S26 and PEVK S170 as CaMKIIδ phosphorylation sites ]. Results show that both sites are phosphorylated by CaMKIIδ. B) Proteins were incubated in kinase buffer (containing Ca 2+ /Calmodulin, and [γ- 32 P]ATP) without (−) and with CaMKIIδ (+). Proteins were then electrophoresed, Coomassie blue stained (top), and exposed to autoradiographic film (bottom). Top: phosphorylation results in incorporation of 32 P in all proteins, but the level varies and is highest in the WT protein. Bottom: Quantitative analysis of 32 P incorporation (normalized to protein loading) shows that the single mutants incorporate only about half the WT amount. The phosphorylation level in the double mutant is ~75% less than in WT PEVK. See text for details. (N=5, * p

Techniques Used: Incubation, Staining, Mutagenesis

37) Product Images from "Farnesoid X Receptor, through the Binding with Steroidogenic Factor 1-responsive Element, Inhibits Aromatase Expression in Tumor Leydig Cells *"

Article Title: Farnesoid X Receptor, through the Binding with Steroidogenic Factor 1-responsive Element, Inhibits Aromatase Expression in Tumor Leydig Cells *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M109.052670

FXR binds to SF-1 site within the aromatase promoter region. A , nuclear extract from R2C cells were incubated with a double-stranded SF-1-specific sequence probe labeled with [γ- 32 P]ATP and subjected to electrophoresis in a 6% polyacrylamide gel
Figure Legend Snippet: FXR binds to SF-1 site within the aromatase promoter region. A , nuclear extract from R2C cells were incubated with a double-stranded SF-1-specific sequence probe labeled with [γ- 32 P]ATP and subjected to electrophoresis in a 6% polyacrylamide gel

Techniques Used: Incubation, Sequencing, Labeling, Electrophoresis

38) Product Images from "The Aurora Kinase in Trypanosoma brucei Plays Distinctive Roles in Metaphase-Anaphase Transition and Cytokinetic Initiation"

Article Title: The Aurora Kinase in Trypanosoma brucei Plays Distinctive Roles in Metaphase-Anaphase Transition and Cytokinetic Initiation

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1000575

Effect of VX-680 on TbAUK1 kinase activity, cell growth and cell cycle progression in the procyclic trypanosomes. (A). Effect of VX-680 on TbAUK1 kinase activity. Recombinant TbAUK1 and histone H3 each fused with GST were purified and incubated with different concentrations of VX-680 (shown at the top) in the presence of γ- 32 P-ATP. The incubation was carried out at room temperature for 60 min. The assay mixtures were separated in SDS-PAGE and analyzed by Phosphor-Imager for the extent of histone H3 phosphorylation. (B). Effect of VX-680 on cell proliferation. Procyclic 427 cell line was cultivated in vitro in the presence of different concentrations of VX-680. Cell density was counted daily and plotted against incubation time. The results were from three independent experiments. (C). Effect of VX-680 on cell cycle progression in a non-synchronous trypanosome cell population. Procyclic 427 cell line was incubated with 10 µM VX-680 for 3 days. Cells were harvested each day after VX-680 treatment for flow cytometry analysis. (D). Effect of VX-680 on cell cycle progression in synchronized trypanosome cells. Procyclic 427 cell line was synchronized in late S-phase with 0.3 mM hydroxyurea for 16 hrs and released. Cells were then incubated with different concentrations of VX-680 for 2 and 4 hrs, respectively and subjected to flow cytometry analysis.
Figure Legend Snippet: Effect of VX-680 on TbAUK1 kinase activity, cell growth and cell cycle progression in the procyclic trypanosomes. (A). Effect of VX-680 on TbAUK1 kinase activity. Recombinant TbAUK1 and histone H3 each fused with GST were purified and incubated with different concentrations of VX-680 (shown at the top) in the presence of γ- 32 P-ATP. The incubation was carried out at room temperature for 60 min. The assay mixtures were separated in SDS-PAGE and analyzed by Phosphor-Imager for the extent of histone H3 phosphorylation. (B). Effect of VX-680 on cell proliferation. Procyclic 427 cell line was cultivated in vitro in the presence of different concentrations of VX-680. Cell density was counted daily and plotted against incubation time. The results were from three independent experiments. (C). Effect of VX-680 on cell cycle progression in a non-synchronous trypanosome cell population. Procyclic 427 cell line was incubated with 10 µM VX-680 for 3 days. Cells were harvested each day after VX-680 treatment for flow cytometry analysis. (D). Effect of VX-680 on cell cycle progression in synchronized trypanosome cells. Procyclic 427 cell line was synchronized in late S-phase with 0.3 mM hydroxyurea for 16 hrs and released. Cells were then incubated with different concentrations of VX-680 for 2 and 4 hrs, respectively and subjected to flow cytometry analysis.

Techniques Used: Activity Assay, Recombinant, Purification, Incubation, SDS Page, In Vitro, Flow Cytometry, Cytometry

39) Product Images from "The ?-Subunit of the SnRK1 Complex Is Phosphorylated by the Plant Cell Death Suppressor Adi3 1The ?-Subunit of the SnRK1 Complex Is Phosphorylated by the Plant Cell Death Suppressor Adi3 1 [C]The ?-Subunit of the SnRK1 Complex Is Phosphorylated by the Plant Cell Death Suppressor Adi3 1 [C] [W]The ?-Subunit of the SnRK1 Complex Is Phosphorylated by the Plant Cell Death Suppressor Adi3 1 [C] [W] [OA]"

Article Title: The ?-Subunit of the SnRK1 Complex Is Phosphorylated by the Plant Cell Death Suppressor Adi3 1The ?-Subunit of the SnRK1 Complex Is Phosphorylated by the Plant Cell Death Suppressor Adi3 1 [C]The ?-Subunit of the SnRK1 Complex Is Phosphorylated by the Plant Cell Death Suppressor Adi3 1 [C] [W]The ?-Subunit of the SnRK1 Complex Is Phosphorylated by the Plant Cell Death Suppressor Adi3 1 [C] [W] [OA]

Journal: Plant Physiology

doi: 10.1104/pp.112.198432

Adi3 phosphorylates Gal83 at Ser-26. A, Adi3 phosphorylates Ser-26 of Gal83. Kinase-active MBP-Adi3 S539D was used to phosphorylate the indicated MBP-Gal83 Ser-to-Ala mutants using [γ- 32 P]ATP in in vitro kinase assays. Quantity One software was
Figure Legend Snippet: Adi3 phosphorylates Gal83 at Ser-26. A, Adi3 phosphorylates Ser-26 of Gal83. Kinase-active MBP-Adi3 S539D was used to phosphorylate the indicated MBP-Gal83 Ser-to-Ala mutants using [γ- 32 P]ATP in in vitro kinase assays. Quantity One software was

Techniques Used: In Vitro, Software

40) Product Images from "Roles of DNA Sequence and Sigma A Factor in Transcription of the vraSR Operon"

Article Title: Roles of DNA Sequence and Sigma A Factor in Transcription of the vraSR Operon

Journal: Journal of Bacteriology

doi: 10.1128/JB.06143-11

DNase I footprints of VraR and VraR-P on the P vraSR R1+1A, -R1+2A, and -R1+3A variants. The bottom strand of each P vraSR variant was end labeled with [γ- 32 P]ATP and incubated with VraR or VraR-P at 2 or 5 μM. After treatment with DNase
Figure Legend Snippet: DNase I footprints of VraR and VraR-P on the P vraSR R1+1A, -R1+2A, and -R1+3A variants. The bottom strand of each P vraSR variant was end labeled with [γ- 32 P]ATP and incubated with VraR or VraR-P at 2 or 5 μM. After treatment with DNase

Techniques Used: Variant Assay, Labeling, Incubation

DNase I footprint of VraR-P on the P vraSR R1DM3 and R2SM1 variants. The top strands of P vraSR , P vraSR R1DM3, and P vraSR R2SM1 were end labeled with [γ- 32 P]ATP. The binding reaction mixtures consisted of VraR-P and 10 ng DNA. After treatment with
Figure Legend Snippet: DNase I footprint of VraR-P on the P vraSR R1DM3 and R2SM1 variants. The top strands of P vraSR , P vraSR R1DM3, and P vraSR R2SM1 were end labeled with [γ- 32 P]ATP. The binding reaction mixtures consisted of VraR-P and 10 ng DNA. After treatment with

Techniques Used: Labeling, Binding Assay

41) Product Images from "A Unique GTP-Dependent Sporulation Sensor Histidine Kinase in Bacillus anthracis ▿ ▿ †"

Article Title: A Unique GTP-Dependent Sporulation Sensor Histidine Kinase in Bacillus anthracis ▿ ▿ †

Journal: Journal of Bacteriology

doi: 10.1128/JB.01184-08

Time course of BA2291 autophosphorylation activity in the presence of [γ- 32 P]GTP alone or with the unlabeled nucleotides GTP, ATP, TTP, and CTP. The in vitro activity assay was carried out with BA2291 (5 μM) purified from E. coli . The
Figure Legend Snippet: Time course of BA2291 autophosphorylation activity in the presence of [γ- 32 P]GTP alone or with the unlabeled nucleotides GTP, ATP, TTP, and CTP. The in vitro activity assay was carried out with BA2291 (5 μM) purified from E. coli . The

Techniques Used: Activity Assay, In Vitro, Purification

42) Product Images from "Intrinsic Protein Kinase Activity in Mitochondrial Oxidative Phosphorylation Complexes"

Article Title: Intrinsic Protein Kinase Activity in Mitochondrial Oxidative Phosphorylation Complexes

Journal: Biochemistry

doi: 10.1021/bi101434x

Reproducibility of a γ- 32 P-ATP-labeled 2D BN/SDS-PAGE gel. Individual panels show replicates from four different animals in different regions of the gel to demonstrate reproducibility. Each panel is labeled for the dominant 32 P-labled protein
Figure Legend Snippet: Reproducibility of a γ- 32 P-ATP-labeled 2D BN/SDS-PAGE gel. Individual panels show replicates from four different animals in different regions of the gel to demonstrate reproducibility. Each panel is labeled for the dominant 32 P-labled protein

Techniques Used: Labeling, SDS Page

Purified Creatine Kinase Radio-labeled in vitro with ATP. Panels A and B show γ- 32 P-ATP and α- 32 P-ATP incorporation into creatine kinase, respectively. For both panels, the Coomassie blue stained image is above the radio-labeled image.
Figure Legend Snippet: Purified Creatine Kinase Radio-labeled in vitro with ATP. Panels A and B show γ- 32 P-ATP and α- 32 P-ATP incorporation into creatine kinase, respectively. For both panels, the Coomassie blue stained image is above the radio-labeled image.

Techniques Used: Purification, Labeling, In Vitro, Staining

Purified Complex V Radio-labeled in vitro with ATP and in Intact Mitochondria. Panels A and B show Complex V labeled in vitro with γ- 32 P-ATP and α- 32 P-ATP, respectively. Panel C shows the labeling pattern of Complex V purified from intact
Figure Legend Snippet: Purified Complex V Radio-labeled in vitro with ATP and in Intact Mitochondria. Panels A and B show Complex V labeled in vitro with γ- 32 P-ATP and α- 32 P-ATP, respectively. Panel C shows the labeling pattern of Complex V purified from intact

Techniques Used: Purification, Labeling, In Vitro

Overlay of a 2D BN/SDS-PAGE following incubation with γ- 32 P-ATP. The Coomassie blue stained gel (A) is colored green in the overlay (B) and the γ- 32 P-ATP-labeled gel (C) is colored red. The overlay image in Panel B applies different contrast
Figure Legend Snippet: Overlay of a 2D BN/SDS-PAGE following incubation with γ- 32 P-ATP. The Coomassie blue stained gel (A) is colored green in the overlay (B) and the γ- 32 P-ATP-labeled gel (C) is colored red. The overlay image in Panel B applies different contrast

Techniques Used: SDS Page, Incubation, Staining, Labeling

Comparison of 2D BN/SDS-PAGE 32 P-labeling after 1D BN-PAGE Incubation with Different 32 P labeled Metabolites. 2D BN/SDS-PAGE gels following incubation of 1D BN-PAGE porcine heart gels with γ- 32 P-ATP (A), α- 32 P-ATP (B), γ- 32 P-GTP
Figure Legend Snippet: Comparison of 2D BN/SDS-PAGE 32 P-labeling after 1D BN-PAGE Incubation with Different 32 P labeled Metabolites. 2D BN/SDS-PAGE gels following incubation of 1D BN-PAGE porcine heart gels with γ- 32 P-ATP (A), α- 32 P-ATP (B), γ- 32 P-GTP

Techniques Used: SDS Page, Labeling, Polyacrylamide Gel Electrophoresis, Incubation

43) Product Images from "Cdc6 degradation requires phosphodegron created by GSK-3 and Cdk1 for SCFCdc4 recognition in Saccharomyces cerevisiae"

Article Title: Cdc6 degradation requires phosphodegron created by GSK-3 and Cdk1 for SCFCdc4 recognition in Saccharomyces cerevisiae

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E14-07-1213

Mck1 phosphorylates the T368 site in Cdc6 after priming by Clb2-Cdk1. (A) To measure Clb2-Cdk1 and Mck1 kinase activities on Cdc6 phosphopeptides, various synthetic peptides of Cdc6 (residues 36–47 or 365–376; shown above) were incubated with purified kinases from asynchronous yeast cultures and [γ- 32 P]ATP. For each kinase, phosphate incorporation was normalized to a control reaction without peptides. Values for the C-terminal peptides represent the average from three independent experiments. Error bars represent SD. (B) Indicated strains were grown in raffinose-containing medium first. Cdc6 expression was induced with galactose for 2 h. Cells were blocked with nocodazole or α-factor for 2 h. Western blotting was performed using anti–phosphoT368 of Cdc6, anti-HA, anti-Pgk1, and anti-Clb2 antibodies, respectively. (C) Indicated strains were grown in raffinose-containing medium first, and then galactose was added to induce Cdc6 expression for 2 h. Cells were blocked with nocodazole for 2 h. Western blotting was performed using anti–phosphoT368 of Cdc6, anti-HA, and anti-Pgk1 antibodies, respectively (left). Band intensity for the phospho-Cdc6-T368 was quantified and normalized to the total amount of Cdc6 (right). The ratio phospho-T368/total Cdc6 in Δclb4 was set as 1. Results are the average of three independent experiments; error bars indicate SD.
Figure Legend Snippet: Mck1 phosphorylates the T368 site in Cdc6 after priming by Clb2-Cdk1. (A) To measure Clb2-Cdk1 and Mck1 kinase activities on Cdc6 phosphopeptides, various synthetic peptides of Cdc6 (residues 36–47 or 365–376; shown above) were incubated with purified kinases from asynchronous yeast cultures and [γ- 32 P]ATP. For each kinase, phosphate incorporation was normalized to a control reaction without peptides. Values for the C-terminal peptides represent the average from three independent experiments. Error bars represent SD. (B) Indicated strains were grown in raffinose-containing medium first. Cdc6 expression was induced with galactose for 2 h. Cells were blocked with nocodazole or α-factor for 2 h. Western blotting was performed using anti–phosphoT368 of Cdc6, anti-HA, anti-Pgk1, and anti-Clb2 antibodies, respectively. (C) Indicated strains were grown in raffinose-containing medium first, and then galactose was added to induce Cdc6 expression for 2 h. Cells were blocked with nocodazole for 2 h. Western blotting was performed using anti–phosphoT368 of Cdc6, anti-HA, and anti-Pgk1 antibodies, respectively (left). Band intensity for the phospho-Cdc6-T368 was quantified and normalized to the total amount of Cdc6 (right). The ratio phospho-T368/total Cdc6 in Δclb4 was set as 1. Results are the average of three independent experiments; error bars indicate SD.

Techniques Used: Incubation, Purification, Expressing, Western Blot

44) Product Images from "FOXO1 Up-Regulates Human L-selectin Expression Through Binding to a Consensus FOXO1 Motif"

Article Title: FOXO1 Up-Regulates Human L-selectin Expression Through Binding to a Consensus FOXO1 Motif

Journal: Gene Regulation and Systems Biology

doi: 10.4137/GRSB.S10343

FOXO1 binds to FOXO1 motif in vitro. Notes: DNA probe containing IRE from APOC3 gene was labeled with γ- 32 P-ATP (lane 1), which was then incubated with nuclear extract from HeLa cells over-expressing FOXO1-3A (lane 2). The protein-DNA complex was then competed with either 10× and 100× cold APOC3 probe (lane 3 and 4, labeled as APOC3), or with 10× and 100× cold probe containing FOXO1 motif from human Sell (lane 5 and 6, labeled as FOXO1), or with 10× and 100× cold probe containing mutated FOXO1 motif from human Sell (lane 7 and 8, labeled as FOXO1m). Free probe was indicated with arrowhead and specific DNA-protein complex was indicated with arrow on the side.
Figure Legend Snippet: FOXO1 binds to FOXO1 motif in vitro. Notes: DNA probe containing IRE from APOC3 gene was labeled with γ- 32 P-ATP (lane 1), which was then incubated with nuclear extract from HeLa cells over-expressing FOXO1-3A (lane 2). The protein-DNA complex was then competed with either 10× and 100× cold APOC3 probe (lane 3 and 4, labeled as APOC3), or with 10× and 100× cold probe containing FOXO1 motif from human Sell (lane 5 and 6, labeled as FOXO1), or with 10× and 100× cold probe containing mutated FOXO1 motif from human Sell (lane 7 and 8, labeled as FOXO1m). Free probe was indicated with arrowhead and specific DNA-protein complex was indicated with arrow on the side.

Techniques Used: In Vitro, Labeling, Incubation, Expressing

45) Product Images from "NME7 is a functional component of the γ-tubulin ring complex"

Article Title: NME7 is a functional component of the γ-tubulin ring complex

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E13-06-0339

NME7 undergoes autophosphorylation. (A) Sequence alignment of the putative kinase domains (NME7A and NME7B) of NME7 with those of other members of the NME family. Asterisks indicate the residues targeted using site-directed mutagenesis. (B, C) Recombinant NME7 (WT) and its mutants were subjected to an autophosphorylation reaction in which either [γ- 32 P]ATP (B) or [γ- 32 P]GTP (C) was used as the phosphate donor. After the reaction, proteins were resolved using SDS–PAGE and examined by means of Coomassie blue staining and autoradiography. H206F, H355F, K173A, and R322A are the mutants.
Figure Legend Snippet: NME7 undergoes autophosphorylation. (A) Sequence alignment of the putative kinase domains (NME7A and NME7B) of NME7 with those of other members of the NME family. Asterisks indicate the residues targeted using site-directed mutagenesis. (B, C) Recombinant NME7 (WT) and its mutants were subjected to an autophosphorylation reaction in which either [γ- 32 P]ATP (B) or [γ- 32 P]GTP (C) was used as the phosphate donor. After the reaction, proteins were resolved using SDS–PAGE and examined by means of Coomassie blue staining and autoradiography. H206F, H355F, K173A, and R322A are the mutants.

Techniques Used: Sequencing, Mutagenesis, Recombinant, SDS Page, Staining, Autoradiography

46) Product Images from "STUDY OF ARACHIDONOYL SPECIFICITY IN TWO ENZYMES OF THE PI-CYCLE"

Article Title: STUDY OF ARACHIDONOYL SPECIFICITY IN TWO ENZYMES OF THE PI-CYCLE

Journal: Journal of molecular biology

doi: 10.1016/j.jmb.2011.03.071

Comparison of inhibition of DGKε by PA. DGK enzymatic activity was measured with 15 mM Triton X-100, 0.1 mM [γ- 32 P]-ATP, and 2 mol % SAG (shown as dark grey bars) or with the addition of either 2 mol % SAPA (light grey bars) or SOPA (white bars). The numbers above the bars show the enzymatic activity of proteins in presence of PA as a percentage of the enzymatic activity in the absence of PA.
Figure Legend Snippet: Comparison of inhibition of DGKε by PA. DGK enzymatic activity was measured with 15 mM Triton X-100, 0.1 mM [γ- 32 P]-ATP, and 2 mol % SAG (shown as dark grey bars) or with the addition of either 2 mol % SAPA (light grey bars) or SOPA (white bars). The numbers above the bars show the enzymatic activity of proteins in presence of PA as a percentage of the enzymatic activity in the absence of PA.

Techniques Used: Inhibition, Activity Assay

47) Product Images from "Sequential Phosphorylation of Smoothened Transduces Graded Hedgehog Signaling"

Article Title: Sequential Phosphorylation of Smoothened Transduces Graded Hedgehog Signaling

Journal: Science signaling

doi: 10.1126/scisignal.2001747

PP1 and PP2A dephosphorylate distinct species of pSmo. ( A and B ) PP1 or PP2A alone incompletely dephosphorylated GFP-pSmo. Hh-induced cl-8– gsmo lysates were treated with increasing amounts of (A) PP1 or (B) PP2A. Western blotting analysis with an antibody against GFP (top panels) detected all forms of GFP-pSmo (box brackets), which were completely removed by CIP. The α-Smo-pS 667 antibody (middle panels) specifically detected PKA-phosphorylated GFP-Smo (GFP-PKA-pSmo). PP1 efficiently dephosphorylated GFP-PKA-pSmo, whereas PP2A treatment enriched for the α-Smo-pS 667 antibody–specific GFP-PKA-pSmo. GFP-PKA-pSmo species treated with PP2A were greatly reduced in abundance by additional PP1 activity (panel B, compare lane 6 with lanes 2 to 4). The Western blot analyzed with the α-Smo-pS 667 antibody (A, middle panel) was overexposed to reveal PKA-pSmo in lysates containing enhanced amounts of pSmo. β-Tubulin served as the loading control. (C and D) PP1 dephosphorylates PKA-phosphorylated Smo. GST-PKA-pSmo labeled with cold ATP was dephosphorylated by PP1, but not by PP2A, as indicated by the decrease in the extent of detection of GST-PKA-pSmo with α-Smo-pS 667 (C). This PP1 response was inhibited by PP1-I2, a specific PP1 inhibitor. Radiolabeled PKA–phosphorylated GST-Smo (GST-PKA- 32 pSmo) was dephosphorylated by PP1, but not by PP2A (D). λ-Phosphatase (λPpase) was used as a positive control in panels C to F. (E) GST-Smo (lane 1) was phosphorylated with PKA alone (lanes 2 and 3) or PKA and CKI (lanes 4 to 8) in the presence of cold ATP. Treating GST-CKI-pSmo with PP2A (lane 5), but not PP1 (lane 6), enriched for the α-Smo-pS 667 –reactive GST-PKA-pSmo, which was reversed by additional PP1 activity (lane 7). (F) PP2A specifically targets CKI consensus modifications. Individual PKA-CKI clusters in GST-Smo were phosphorylated by PKA in the presence of ATP followed by CKI-mediated phosphorylation in the presence of [γ- 32 P]ATP (lanes 1, 3, and 5), resulting in selective incorporation of [γ- 32 P]ATP only at CKI consensus serines within a single cluster, as schematically shown. PP2A removed CKI phosphorylation–specific 32 P-phosphates from individual PKA-CKI clusters (lanes 2, 4, and 6).
Figure Legend Snippet: PP1 and PP2A dephosphorylate distinct species of pSmo. ( A and B ) PP1 or PP2A alone incompletely dephosphorylated GFP-pSmo. Hh-induced cl-8– gsmo lysates were treated with increasing amounts of (A) PP1 or (B) PP2A. Western blotting analysis with an antibody against GFP (top panels) detected all forms of GFP-pSmo (box brackets), which were completely removed by CIP. The α-Smo-pS 667 antibody (middle panels) specifically detected PKA-phosphorylated GFP-Smo (GFP-PKA-pSmo). PP1 efficiently dephosphorylated GFP-PKA-pSmo, whereas PP2A treatment enriched for the α-Smo-pS 667 antibody–specific GFP-PKA-pSmo. GFP-PKA-pSmo species treated with PP2A were greatly reduced in abundance by additional PP1 activity (panel B, compare lane 6 with lanes 2 to 4). The Western blot analyzed with the α-Smo-pS 667 antibody (A, middle panel) was overexposed to reveal PKA-pSmo in lysates containing enhanced amounts of pSmo. β-Tubulin served as the loading control. (C and D) PP1 dephosphorylates PKA-phosphorylated Smo. GST-PKA-pSmo labeled with cold ATP was dephosphorylated by PP1, but not by PP2A, as indicated by the decrease in the extent of detection of GST-PKA-pSmo with α-Smo-pS 667 (C). This PP1 response was inhibited by PP1-I2, a specific PP1 inhibitor. Radiolabeled PKA–phosphorylated GST-Smo (GST-PKA- 32 pSmo) was dephosphorylated by PP1, but not by PP2A (D). λ-Phosphatase (λPpase) was used as a positive control in panels C to F. (E) GST-Smo (lane 1) was phosphorylated with PKA alone (lanes 2 and 3) or PKA and CKI (lanes 4 to 8) in the presence of cold ATP. Treating GST-CKI-pSmo with PP2A (lane 5), but not PP1 (lane 6), enriched for the α-Smo-pS 667 –reactive GST-PKA-pSmo, which was reversed by additional PP1 activity (lane 7). (F) PP2A specifically targets CKI consensus modifications. Individual PKA-CKI clusters in GST-Smo were phosphorylated by PKA in the presence of ATP followed by CKI-mediated phosphorylation in the presence of [γ- 32 P]ATP (lanes 1, 3, and 5), resulting in selective incorporation of [γ- 32 P]ATP only at CKI consensus serines within a single cluster, as schematically shown. PP2A removed CKI phosphorylation–specific 32 P-phosphates from individual PKA-CKI clusters (lanes 2, 4, and 6).

Techniques Used: Western Blot, Activity Assay, Labeling, Positive Control

48) Product Images from "C-terminal Src Kinase (Csk)-mediated Phosphorylation of Eukaryotic Elongation Factor 2 (eEF2) Promotes Proteolytic Cleavage and Nuclear Translocation of eEF2 *"

Article Title: C-terminal Src Kinase (Csk)-mediated Phosphorylation of Eukaryotic Elongation Factor 2 (eEF2) Promotes Proteolytic Cleavage and Nuclear Translocation of eEF2 *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M113.546481

Identification of new protein substrates for tyrosine kinase Csk. A and B , two-dimensional gel electrophoresis analysis of Csk −/− MEF cell lysates in the presence of [γ- 32 P]ATP alone ( A ) or in the presence of purified Csk and [γ- 32 P]ATP ( B ). C, phosphorylation of purified eEF2 by purified Csk in vitro. D, different concentrations of purified Csk (0, 50, and 100 ng) phosphorylate purified eEF2 in vitro , and anti-phosphotyrosine and anti-N-terminal eEF2 antibodies were used for blotting. E , identification of phosphorylated tyrosine residues on eEF2 by Csk. Mutant and wild-type eEF2 plasmids were tagged with Myc and transfected into HEK293T cells with or without Csk plasmid. Anti-Myc antibody was used for immunoprecipitation ( IP ). The blot was probed with either anti-phosphotyrosine monoclonal antibody ( top panel ) or with anti-Myc antibody ( bottom panel ). F, co-immunoprecipitation of Csk and eEF2. Plasmids of pcDNA3.0 and FLAG-Csk were transfected into HEK293T cells. Left panel, blots showed that anti-C-terminal eEF2 antibody co-immunoprecipitated FLAG-tagged Csk; middle panel, anti-FLAG antibody (for FLAG-tagged Csk) also co-immunoprecipitated eEF2. Input, whole cell lysates from cells transfected with FLAG-tagged Csk. Right panel, top, Western blot ( WB ) with anti-FLAG antibody shows the expression of FLAG-tagged Csk. Bottom, Western blot with anti-actin antibody shows similar amounts of total cell lysates were used in all immunoprecipitation experiments. G, interaction of endogenous eEF2 and Csk. Left panel, anti-C-terminal eEF2 antibody that targets endogenous eEF2 in MEF cells could co-immunoprecipitate endogenous Csk. Right panel, Western blot with anti-Csk antibody shows the absence of Csk expression in Csk −/− MEF cells ( top ), and anti-C-terminal eEF2 antibody shows similar levels of eEF2. Data are representative of three independent experiments.
Figure Legend Snippet: Identification of new protein substrates for tyrosine kinase Csk. A and B , two-dimensional gel electrophoresis analysis of Csk −/− MEF cell lysates in the presence of [γ- 32 P]ATP alone ( A ) or in the presence of purified Csk and [γ- 32 P]ATP ( B ). C, phosphorylation of purified eEF2 by purified Csk in vitro. D, different concentrations of purified Csk (0, 50, and 100 ng) phosphorylate purified eEF2 in vitro , and anti-phosphotyrosine and anti-N-terminal eEF2 antibodies were used for blotting. E , identification of phosphorylated tyrosine residues on eEF2 by Csk. Mutant and wild-type eEF2 plasmids were tagged with Myc and transfected into HEK293T cells with or without Csk plasmid. Anti-Myc antibody was used for immunoprecipitation ( IP ). The blot was probed with either anti-phosphotyrosine monoclonal antibody ( top panel ) or with anti-Myc antibody ( bottom panel ). F, co-immunoprecipitation of Csk and eEF2. Plasmids of pcDNA3.0 and FLAG-Csk were transfected into HEK293T cells. Left panel, blots showed that anti-C-terminal eEF2 antibody co-immunoprecipitated FLAG-tagged Csk; middle panel, anti-FLAG antibody (for FLAG-tagged Csk) also co-immunoprecipitated eEF2. Input, whole cell lysates from cells transfected with FLAG-tagged Csk. Right panel, top, Western blot ( WB ) with anti-FLAG antibody shows the expression of FLAG-tagged Csk. Bottom, Western blot with anti-actin antibody shows similar amounts of total cell lysates were used in all immunoprecipitation experiments. G, interaction of endogenous eEF2 and Csk. Left panel, anti-C-terminal eEF2 antibody that targets endogenous eEF2 in MEF cells could co-immunoprecipitate endogenous Csk. Right panel, Western blot with anti-Csk antibody shows the absence of Csk expression in Csk −/− MEF cells ( top ), and anti-C-terminal eEF2 antibody shows similar levels of eEF2. Data are representative of three independent experiments.

Techniques Used: Two-Dimensional Gel Electrophoresis, Electrophoresis, Purification, In Vitro, Mutagenesis, Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Expressing

49) Product Images from "A New Class of Quorum Quenching Molecules from Staphylococcus Species Affects Communication and Growth of Gram-Negative Bacteria"

Article Title: A New Class of Quorum Quenching Molecules from Staphylococcus Species Affects Communication and Growth of Gram-Negative Bacteria

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1003654

Yayurea A and B are perceived by V. harveyi LuxN receptor. ( A ) Schematic representation of the QS phosphorelay in V. harveyi . In the absence of autoinducers (HAI-1, AI-2 and CAI-1) at low cell density, each of the three receptors, LuxN, LuxQ and CqsS, respectively, autophosphorylates at a conserved histidine of their histidine kinase domain (HK). The phosphoryl group is first transferred to the receiver domain (Rec) of the receptor kinase and then to the HPt protein LuxU. LuxP is a periplasmic binding protein. P denotes phosphorylation sites. Upon perception of the autoinducers at high cell density, autophosphorylation of the receptors and the subsequent phosphosphrylation cascade is inhibited. Yayurea A and B stimulated the phosphorylation of the cascade via LuxN. ( B ) LuxPQ and CqsS mediated phosphorylation of LuxU in the presence of yayurea A and B. ( C ) LuxN mediated phosphorylation of LuxU in the presence of yayurea A and B and HAI-1. LuxQ, CqsS, LuxN-bearing membrane vesicles and LuxU, were incubated with 100 µM [γ- 32 P] ATP. The effect of yayurea A, B and HAI-1 ( C ) on the initial rate of LuxU phosphorylation was evaluated. Each reaction was sampled and stopped at two different time points: after 1 and 10 minutes. Final concentrations were 20 µM for HAI-1, 1.1 mM for yayurea A and 1.3 mM yayurea B, which reflects the in vivo situation. Absence of HAI-1 or yayurea A or B is indicated by “−” and presence by “+”.
Figure Legend Snippet: Yayurea A and B are perceived by V. harveyi LuxN receptor. ( A ) Schematic representation of the QS phosphorelay in V. harveyi . In the absence of autoinducers (HAI-1, AI-2 and CAI-1) at low cell density, each of the three receptors, LuxN, LuxQ and CqsS, respectively, autophosphorylates at a conserved histidine of their histidine kinase domain (HK). The phosphoryl group is first transferred to the receiver domain (Rec) of the receptor kinase and then to the HPt protein LuxU. LuxP is a periplasmic binding protein. P denotes phosphorylation sites. Upon perception of the autoinducers at high cell density, autophosphorylation of the receptors and the subsequent phosphosphrylation cascade is inhibited. Yayurea A and B stimulated the phosphorylation of the cascade via LuxN. ( B ) LuxPQ and CqsS mediated phosphorylation of LuxU in the presence of yayurea A and B. ( C ) LuxN mediated phosphorylation of LuxU in the presence of yayurea A and B and HAI-1. LuxQ, CqsS, LuxN-bearing membrane vesicles and LuxU, were incubated with 100 µM [γ- 32 P] ATP. The effect of yayurea A, B and HAI-1 ( C ) on the initial rate of LuxU phosphorylation was evaluated. Each reaction was sampled and stopped at two different time points: after 1 and 10 minutes. Final concentrations were 20 µM for HAI-1, 1.1 mM for yayurea A and 1.3 mM yayurea B, which reflects the in vivo situation. Absence of HAI-1 or yayurea A or B is indicated by “−” and presence by “+”.

Techniques Used: Binding Assay, Incubation, In Vivo

50) Product Images from "MOLECULAR MECHANISM OF HUMAN NRF2 ACTIVATION AND DEGRADATION: ROLE OF SEQUENTIAL PHOSPHORYLATION BY PROTEIN KINASE CK2"

Article Title: MOLECULAR MECHANISM OF HUMAN NRF2 ACTIVATION AND DEGRADATION: ROLE OF SEQUENTIAL PHOSPHORYLATION BY PROTEIN KINASE CK2

Journal:

doi: 10.1016/j.freeradbiomed.2007.03.001

Human Nrf2 is a substrate of CK2. Recombinant human Nrf2 (A) and Nrf2 immunoprecipitation products from the nuclear extracts of acute As-exposed HaCaT cells (B) were incubated with recombinant human CK2 (1000 U/ml) in CK2 buffer with [γ- 32 P]ATP
Figure Legend Snippet: Human Nrf2 is a substrate of CK2. Recombinant human Nrf2 (A) and Nrf2 immunoprecipitation products from the nuclear extracts of acute As-exposed HaCaT cells (B) were incubated with recombinant human CK2 (1000 U/ml) in CK2 buffer with [γ- 32 P]ATP

Techniques Used: Recombinant, Immunoprecipitation, Incubation

51) Product Images from "The B-Cell-Specific Transcription Factor and Master Regulator Pax5 Promotes Epstein-Barr Virus Latency by Negatively Regulating the Viral Immediate Early Protein BZLF1"

Article Title: The B-Cell-Specific Transcription Factor and Master Regulator Pax5 Promotes Epstein-Barr Virus Latency by Negatively Regulating the Viral Immediate Early Protein BZLF1

Journal: Journal of Virology

doi: 10.1128/JVI.00546-13

A Pax5 double mutant is DNA binding deficient and interacts with Z in vitro . (A) For the left panel, a gel-shift assay was performed using in vitro -translated wild-type or double-mutant Pax5 V26G/P80R (labeled DM) and [γ- 32 P]ATP-labeled CD19 oligonucleotide
Figure Legend Snippet: A Pax5 double mutant is DNA binding deficient and interacts with Z in vitro . (A) For the left panel, a gel-shift assay was performed using in vitro -translated wild-type or double-mutant Pax5 V26G/P80R (labeled DM) and [γ- 32 P]ATP-labeled CD19 oligonucleotide

Techniques Used: Mutagenesis, Binding Assay, In Vitro, Electrophoretic Mobility Shift Assay, Labeling

52) Product Images from "Blue light and CO2 signals converge to regulate light-induced stomatal opening"

Article Title: Blue light and CO2 signals converge to regulate light-induced stomatal opening

Journal: Nature Communications

doi: 10.1038/s41467-017-01237-5

Phosphorylation of CBC1 by phototropin1 in a BL-dependent manner. a BL-dependent mobility shift of CBC1. GCPs were illuminated with light as described in Fig. 1a . Samples were obtained 1 min after the start of the BL pulse. Immunoblot analysis of CBC1 was performed after electrophoresis on Phos-tag SDS-PAGE that contained 10% acrylamide, 20 μM Phos-tag, and 40 μM MnCl 2 . For CBC2, 40 μM Phos-tag and 80 μM MnCl 2 were included in the gel. b Phototropin-mediated phosphorylation of CBC1. CBC1 detection was done as described in a . c Pull-down assays of CBC1 and CBC2 by phot1. FLAG-phot1 and His-CBCs were synthesized in vitro transcription/translation. d BiFC assays of CBCs and phot1. CBCs-nYFP and phot1-cYFP were co-transfected to MCPs. White bars represent 20 μm. e Phosphorylation of kinase-dead CBC1 (D271N) and CBC2 (D245N) by P1C using [γ- 32 P] ATP. Phosphorylation assays were performed in 30 μl reaction mixtures that contained 50 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 , 3.3 μM ATP, 20 μCi of [γ- 32 P] ATP, and purified proteins. The reactions proceeded for 2 h at 15 °C. GST-tagged P1C or kinase-dead P1C (D806N) were included at 2.5 μg, and CBC1 (D271N) and CBC2 (D245N) were added at 1.2 μg. These GST proteins were expressed in E. coli and purified with glutathione-Sepharose beads. f Phosphorylation of kinase-dead CBC1 (D271N) and CBC1 (D271N S43A S45A) by P1C. Measurement was done as same as e . g Mobility of CBC1-GFP and CBC1 (S43A S45A)-GFP expressed in guard cells was determined as a , except the acrylamide concentration was used at 6%. The cbc1 cbc2 double mutant was transformed with CBC-GFP or CBC1 ( S43A S45A ) -GFP and GCPs were prepared from these transgenic plants. h Pull-down assays of His-CBC1 and His-CBC2 by FLAG-tagged CBC1 and CBC2. Tagged proteins were synthesized by in vitro transcription/translation. i BL-dependent phosphorylation of CBC1 in the blus1-3 mutant. CBC1 detection was done as described in a
Figure Legend Snippet: Phosphorylation of CBC1 by phototropin1 in a BL-dependent manner. a BL-dependent mobility shift of CBC1. GCPs were illuminated with light as described in Fig. 1a . Samples were obtained 1 min after the start of the BL pulse. Immunoblot analysis of CBC1 was performed after electrophoresis on Phos-tag SDS-PAGE that contained 10% acrylamide, 20 μM Phos-tag, and 40 μM MnCl 2 . For CBC2, 40 μM Phos-tag and 80 μM MnCl 2 were included in the gel. b Phototropin-mediated phosphorylation of CBC1. CBC1 detection was done as described in a . c Pull-down assays of CBC1 and CBC2 by phot1. FLAG-phot1 and His-CBCs were synthesized in vitro transcription/translation. d BiFC assays of CBCs and phot1. CBCs-nYFP and phot1-cYFP were co-transfected to MCPs. White bars represent 20 μm. e Phosphorylation of kinase-dead CBC1 (D271N) and CBC2 (D245N) by P1C using [γ- 32 P] ATP. Phosphorylation assays were performed in 30 μl reaction mixtures that contained 50 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 , 3.3 μM ATP, 20 μCi of [γ- 32 P] ATP, and purified proteins. The reactions proceeded for 2 h at 15 °C. GST-tagged P1C or kinase-dead P1C (D806N) were included at 2.5 μg, and CBC1 (D271N) and CBC2 (D245N) were added at 1.2 μg. These GST proteins were expressed in E. coli and purified with glutathione-Sepharose beads. f Phosphorylation of kinase-dead CBC1 (D271N) and CBC1 (D271N S43A S45A) by P1C. Measurement was done as same as e . g Mobility of CBC1-GFP and CBC1 (S43A S45A)-GFP expressed in guard cells was determined as a , except the acrylamide concentration was used at 6%. The cbc1 cbc2 double mutant was transformed with CBC-GFP or CBC1 ( S43A S45A ) -GFP and GCPs were prepared from these transgenic plants. h Pull-down assays of His-CBC1 and His-CBC2 by FLAG-tagged CBC1 and CBC2. Tagged proteins were synthesized by in vitro transcription/translation. i BL-dependent phosphorylation of CBC1 in the blus1-3 mutant. CBC1 detection was done as described in a

Techniques Used: Mobility Shift, Electrophoresis, SDS Page, Synthesized, In Vitro, Bimolecular Fluorescence Complementation Assay, Transfection, Purification, Concentration Assay, Mutagenesis, Transformation Assay, Transgenic Assay

53) Product Images from "Analysis of small RNA in fission yeast; centromeric siRNAs are potentially generated through a structured RNA"

Article Title: Analysis of small RNA in fission yeast; centromeric siRNAs are potentially generated through a structured RNA

Journal: The EMBO Journal

doi: 10.1038/emboj.2009.351

Secondary structure determination of RevCen. ( A ) Secondary structure probing of in vitro transcribed, [γ- 32 P]ATP 5′-end-labelled RevCen RNA analysed on polyacrylamide gels. Partial RNA cleavages were performed as described in Materials and methods section. An OH ladder and a T1 ladder were used to assess cleavage positions. The positions of G residues are marked on the right. ( B ) Refined prediction of the RevCen secondary structure using the Mfold software using constraints from structural probing data in ( A ). The siRNAs VII and VIII are indicated in light and dark orange. The grey shade indicates the part of RevCen , which could not be resolved. Nucleotides are highlighted with different colours to show probe-dependent cleavage, as indicated in the colour key. ( C ) Cleavage of RevCen by human Dicer in vitro . RNA fragments ranging from 21–33 nt were formed when treating RevCen with recombinant human Dicer. RNA was labeled after incubation. The increasing dose of Dicer is indicated. Incubation times were as follows: 5 min for lanes 1 and 4; 30 min for lanes 2 and 5; 2 h for lanes 3, 6 and 7. Concentrations of Dicer: lane 7: 0 units; lanes 1–3: 0.1 units and lanes 4–6: 1.0 units.
Figure Legend Snippet: Secondary structure determination of RevCen. ( A ) Secondary structure probing of in vitro transcribed, [γ- 32 P]ATP 5′-end-labelled RevCen RNA analysed on polyacrylamide gels. Partial RNA cleavages were performed as described in Materials and methods section. An OH ladder and a T1 ladder were used to assess cleavage positions. The positions of G residues are marked on the right. ( B ) Refined prediction of the RevCen secondary structure using the Mfold software using constraints from structural probing data in ( A ). The siRNAs VII and VIII are indicated in light and dark orange. The grey shade indicates the part of RevCen , which could not be resolved. Nucleotides are highlighted with different colours to show probe-dependent cleavage, as indicated in the colour key. ( C ) Cleavage of RevCen by human Dicer in vitro . RNA fragments ranging from 21–33 nt were formed when treating RevCen with recombinant human Dicer. RNA was labeled after incubation. The increasing dose of Dicer is indicated. Incubation times were as follows: 5 min for lanes 1 and 4; 30 min for lanes 2 and 5; 2 h for lanes 3, 6 and 7. Concentrations of Dicer: lane 7: 0 units; lanes 1–3: 0.1 units and lanes 4–6: 1.0 units.

Techniques Used: In Vitro, Software, Recombinant, Labeling, Incubation

54) Product Images from "Structure and mechanism of a bacterial t6A biosynthesis system"

Article Title: Structure and mechanism of a bacterial t6A biosynthesis system

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkx1300

TsaE is required for multiple turnover of the t 6 A biosynthesis reaction. ( A ) Standard radiochemical t 6 A assays measuring the incorporation of [ 14 C]-threonine into tRNA. Assays contained Thermotoga maritima TsaC2 together with TsaB, TsaD and TsaE (open squares) or TsaB and TsaD (filled squares). ( B ) Native agarose gel-shift analysis showing the association of wild-type TsaE and mutants TsaE E108A and TsaE T42A with TsaB 2 D 2 in the absence of nucleotides. ( C ) TLC radiogram of the ATP hydrolysis reactions (generating ADP and P i ) catalyzed by wild-type TsaE, and mutants TsaE E108A and TsaE T42A , in the absence and presence of TsaB 2 D 2 . [γ- 32 P] ATP and the radiolabeled hydrolysis product γ-phosphate are indicated. ( D ) Standard radiochemical t 6 A assays measuring the incorporation of [ 14 C]-threonine into tRNA. Assays contained T. maritima TsaC2, TsaB and TsaD either alone (filled squares), or with added TsaE E108A (open squares) or TsaE T42A (open triangles). ( E ) Radiochemical t 6 A assays ([ 14 C]threonine) analyzed by native PAGE. Reaction assays were carried out as described in the ‘Materials and Methods’ section, and contained 8 μM each of TsaC2, TsaB and TsaD ([TsaB 2 D 2 ] therefore 4 μM). The [tRNA] in assays analyzed in lanes 1, 3 and 5 was 40 μM, while the [tRNA] in the assay analyzed in lane 4 was 3.75 μM. Lane 1 . Standard assay (assay contains TsaB, TsaD and TsaE). Lane 2 . Control lane—assay lacking tRNA. Lane 3 . Assay contains TsaB and TsaD. Lane 4 . Assay contains TsaB and TsaD (low tRNA). Lane 5 . Assay contains TsaB, TsaD and TsaE E108A . Lane 6 . Control lane—[ 14 C]-labeled t 6 A-modified tRNA.
Figure Legend Snippet: TsaE is required for multiple turnover of the t 6 A biosynthesis reaction. ( A ) Standard radiochemical t 6 A assays measuring the incorporation of [ 14 C]-threonine into tRNA. Assays contained Thermotoga maritima TsaC2 together with TsaB, TsaD and TsaE (open squares) or TsaB and TsaD (filled squares). ( B ) Native agarose gel-shift analysis showing the association of wild-type TsaE and mutants TsaE E108A and TsaE T42A with TsaB 2 D 2 in the absence of nucleotides. ( C ) TLC radiogram of the ATP hydrolysis reactions (generating ADP and P i ) catalyzed by wild-type TsaE, and mutants TsaE E108A and TsaE T42A , in the absence and presence of TsaB 2 D 2 . [γ- 32 P] ATP and the radiolabeled hydrolysis product γ-phosphate are indicated. ( D ) Standard radiochemical t 6 A assays measuring the incorporation of [ 14 C]-threonine into tRNA. Assays contained T. maritima TsaC2, TsaB and TsaD either alone (filled squares), or with added TsaE E108A (open squares) or TsaE T42A (open triangles). ( E ) Radiochemical t 6 A assays ([ 14 C]threonine) analyzed by native PAGE. Reaction assays were carried out as described in the ‘Materials and Methods’ section, and contained 8 μM each of TsaC2, TsaB and TsaD ([TsaB 2 D 2 ] therefore 4 μM). The [tRNA] in assays analyzed in lanes 1, 3 and 5 was 40 μM, while the [tRNA] in the assay analyzed in lane 4 was 3.75 μM. Lane 1 . Standard assay (assay contains TsaB, TsaD and TsaE). Lane 2 . Control lane—assay lacking tRNA. Lane 3 . Assay contains TsaB and TsaD. Lane 4 . Assay contains TsaB and TsaD (low tRNA). Lane 5 . Assay contains TsaB, TsaD and TsaE E108A . Lane 6 . Control lane—[ 14 C]-labeled t 6 A-modified tRNA.

Techniques Used: Agarose Gel Electrophoresis, Thin Layer Chromatography, Clear Native PAGE, Labeling, Modification

55) Product Images from "Essential Role of Sphingosine Kinase 2 in the Regulation of Cargo Contents in the Exosomes from K562 Cells"

Article Title: Essential Role of Sphingosine Kinase 2 in the Regulation of Cargo Contents in the Exosomes from K562 Cells

Journal: Kobe Journal of Medical Sciences

doi:

Effect of DMS on in vitro SphK2 activity Affinity-purified recombinant Sphk2 expressed in insect cells was assayed for its ability to phosphorylate sphingosine (100 μM (A, C, D), 0.2 μM (B, E) or absence (C) ) using [γ- 32 P]ATP in the presence of various concentrations of DMS as specified. After termination of the reaction, radio-labeled S1P was separated by thin-layer chromatography followed by autoradiography. One of the representative data from three independent experiments is shown (A). The bands corresponding to S1P were quantitated using a Fujix Bio-Imaging Analyzer. Data are means ± s.e.m. from 3 independent experiments (B).
Figure Legend Snippet: Effect of DMS on in vitro SphK2 activity Affinity-purified recombinant Sphk2 expressed in insect cells was assayed for its ability to phosphorylate sphingosine (100 μM (A, C, D), 0.2 μM (B, E) or absence (C) ) using [γ- 32 P]ATP in the presence of various concentrations of DMS as specified. After termination of the reaction, radio-labeled S1P was separated by thin-layer chromatography followed by autoradiography. One of the representative data from three independent experiments is shown (A). The bands corresponding to S1P were quantitated using a Fujix Bio-Imaging Analyzer. Data are means ± s.e.m. from 3 independent experiments (B).

Techniques Used: In Vitro, Activity Assay, Affinity Purification, Labeling, Thin Layer Chromatography, Autoradiography, Imaging

56) 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

57) Product Images from "Structural Mechanisms of mTORC1 activation by RHEB and inhibition by PRAS40"

Article Title: Structural Mechanisms of mTORC1 activation by RHEB and inhibition by PRAS40

Journal: Nature

doi: 10.1038/nature25023

RHEB-induced conformational change in mTORC1 a , Stereo view of the cryo-EM density from the 3.4 Å RHEB-mTORC1 reconstruction, showing the RHEB-mTOR interface in the same orientation and coloring as in Figure 4c . The RHEB-interacting segments of mTOR are nα3 to nα7 of N-heat, mα2-mα4 of M-heat and fα2-fα3 of FAT. The majority of the contacts made by RHEB are from its switch I and switch II regions, with a small number of additional contacts to N-heat and M-heat contributed by the nearby segments of residues 5 to 7 and 106 to 111. b , Stereo view of cryo-EM density from RHEB-mTORC1 (sand) and the 3.0 Å apo-mTORC1 (green), with the two structures and maps superimposed on the C lobes as in Figure 5d . c , AMPPNP (orange) cryo-EM density of apo-mTORC1. d , Steady state kinetic analysis of mTORC1 phosphorylation of intact 4EBP1 in the presence of 250 μM RHEB-GTPγS. Reactions quantified by 32 P incorporation and plotted as velocity over enzyme concentration (means as dashes and values from two independent experiments as filled circles). The K M and k cat values, calculated by non-linear regression fitting of the data, and simulated curves are also shown. Note that in contrast to the reaction in the absence of RHEB shown in Extended Data Figure 4e , the curve of reaction velocity versus 4EBP1 concentration obeys Michaelis-Menten kinetics. e , RHEB-GTPγS activation of 4EBP1 phosphorylation by mTORC1 under single-turnover conditions. A master mix of excess mTORC1 (500 nM) over 4EBP1 substrate (100 nM) was incubated with 250 μM RHEB-GTPγS or 250 μM RHEB-GDP in the standard kinase buffer on ice for 5 minutes. Reactions were started by the addition of a mixture of cold ATP (50 μM final) and [γ - 32 P] ATP (8 μCi per reaction time point). The reactions were done on ice to slow down the reaction. At indicated time points, an aliquot of the reaction was drawn, stopped, and analyzed as described in Methods. The experiment was repeated three times with very similar results. f , ATP steady-state kinetic parameters of ATP hydrolysis by mTORC1 in the presence of 250 μM RHEB-GDP (left, blue plot) or RHEB-GTPγS (right, red plot). Reactions quantified by 32 P incorporation as in d . Graph shows means as dashes and values from three independent experiments with the indicated markers and colors. The steady-state kinetic constants of the RHEB-GDP containing reaction are approximate owing to the weak signal of these reactions. g , As expected, RHEB-GTPγS did not activate the truncated mTOR ΔN -mLST8 complex phosphorylating 4EBP1 or S6K1 367–404 (10 μM both; mTOR ΔN at 20 and 30 nM, respectively) (left panel; experiments were repeated two times with very similar results). mTOR ΔN -mLST8 has an intermediate k cat of 0.66 s −1 ( Extended Data Fig. 1b ) compared to the 0.09 and 2.9 s −1 k cat values of apo-mTORC1 and RHEB-mTORC1, respectively ( Fig. 5g ). mTOR ΔN -mLST8 has a distinct FAT conformation likely due to the absence of N-heat. Right panel shows superposition of the FAT plus kinase domain portions of inactive apo-mTORC1 on the crystal structure of mTOR ΔN -mLST8 done by aligning their C lobes. Apo-mTORC1 is in green and mTOR ΔN is colored blue for FAT, yellow for N-lobe and pink for C-lobe. The rotation axes (red lines) are numbered according to the hinges of Figure 5c . Compared to the inactive to active transition, the comparison of the mTOR ΔN FAT conformation to that of the inactive state exhibits bigger changes around the major hinge with a rotation in the opposite direction and a different rotation axis far from the hinge axis (labeled “1”). The rotations around the two minor hinges are comparably modest although distinct, with the rotation axes nearly orthogonal to those of the inactive to active transition. h , Autoradiogram showing activation of mTORC1 phosphorylating 4EBP1 (10 μM) by RHEB-GTPγS, repeated three times. Gel quantification is shown in Figure 5b . i , Steady-state kinetic analysis of mTORC1 phosphorylating S6K1 367–404 in presence of 250 μM RHEB-GDP (top row) or RHEB-GTPγS (bottom row). 32 P incorporation data is plotted as velocity over enzyme concentration in Figure 5g ( n =3).
Figure Legend Snippet: RHEB-induced conformational change in mTORC1 a , Stereo view of the cryo-EM density from the 3.4 Å RHEB-mTORC1 reconstruction, showing the RHEB-mTOR interface in the same orientation and coloring as in Figure 4c . The RHEB-interacting segments of mTOR are nα3 to nα7 of N-heat, mα2-mα4 of M-heat and fα2-fα3 of FAT. The majority of the contacts made by RHEB are from its switch I and switch II regions, with a small number of additional contacts to N-heat and M-heat contributed by the nearby segments of residues 5 to 7 and 106 to 111. b , Stereo view of cryo-EM density from RHEB-mTORC1 (sand) and the 3.0 Å apo-mTORC1 (green), with the two structures and maps superimposed on the C lobes as in Figure 5d . c , AMPPNP (orange) cryo-EM density of apo-mTORC1. d , Steady state kinetic analysis of mTORC1 phosphorylation of intact 4EBP1 in the presence of 250 μM RHEB-GTPγS. Reactions quantified by 32 P incorporation and plotted as velocity over enzyme concentration (means as dashes and values from two independent experiments as filled circles). The K M and k cat values, calculated by non-linear regression fitting of the data, and simulated curves are also shown. Note that in contrast to the reaction in the absence of RHEB shown in Extended Data Figure 4e , the curve of reaction velocity versus 4EBP1 concentration obeys Michaelis-Menten kinetics. e , RHEB-GTPγS activation of 4EBP1 phosphorylation by mTORC1 under single-turnover conditions. A master mix of excess mTORC1 (500 nM) over 4EBP1 substrate (100 nM) was incubated with 250 μM RHEB-GTPγS or 250 μM RHEB-GDP in the standard kinase buffer on ice for 5 minutes. Reactions were started by the addition of a mixture of cold ATP (50 μM final) and [γ - 32 P] ATP (8 μCi per reaction time point). The reactions were done on ice to slow down the reaction. At indicated time points, an aliquot of the reaction was drawn, stopped, and analyzed as described in Methods. The experiment was repeated three times with very similar results. f , ATP steady-state kinetic parameters of ATP hydrolysis by mTORC1 in the presence of 250 μM RHEB-GDP (left, blue plot) or RHEB-GTPγS (right, red plot). Reactions quantified by 32 P incorporation as in d . Graph shows means as dashes and values from three independent experiments with the indicated markers and colors. The steady-state kinetic constants of the RHEB-GDP containing reaction are approximate owing to the weak signal of these reactions. g , As expected, RHEB-GTPγS did not activate the truncated mTOR ΔN -mLST8 complex phosphorylating 4EBP1 or S6K1 367–404 (10 μM both; mTOR ΔN at 20 and 30 nM, respectively) (left panel; experiments were repeated two times with very similar results). mTOR ΔN -mLST8 has an intermediate k cat of 0.66 s −1 ( Extended Data Fig. 1b ) compared to the 0.09 and 2.9 s −1 k cat values of apo-mTORC1 and RHEB-mTORC1, respectively ( Fig. 5g ). mTOR ΔN -mLST8 has a distinct FAT conformation likely due to the absence of N-heat. Right panel shows superposition of the FAT plus kinase domain portions of inactive apo-mTORC1 on the crystal structure of mTOR ΔN -mLST8 done by aligning their C lobes. Apo-mTORC1 is in green and mTOR ΔN is colored blue for FAT, yellow for N-lobe and pink for C-lobe. The rotation axes (red lines) are numbered according to the hinges of Figure 5c . Compared to the inactive to active transition, the comparison of the mTOR ΔN FAT conformation to that of the inactive state exhibits bigger changes around the major hinge with a rotation in the opposite direction and a different rotation axis far from the hinge axis (labeled “1”). The rotations around the two minor hinges are comparably modest although distinct, with the rotation axes nearly orthogonal to those of the inactive to active transition. h , Autoradiogram showing activation of mTORC1 phosphorylating 4EBP1 (10 μM) by RHEB-GTPγS, repeated three times. Gel quantification is shown in Figure 5b . i , Steady-state kinetic analysis of mTORC1 phosphorylating S6K1 367–404 in presence of 250 μM RHEB-GDP (top row) or RHEB-GTPγS (bottom row). 32 P incorporation data is plotted as velocity over enzyme concentration in Figure 5g ( n =3).

Techniques Used: Concentration Assay, Activation Assay, Incubation, Labeling

58) Product Images from "Cross-regulation between Aurora B and Citron kinase controls midbody architecture in cytokinesis"

Article Title: Cross-regulation between Aurora B and Citron kinase controls midbody architecture in cytokinesis

Journal: Open Biology

doi: 10.1098/rsob.160019

Aurora B phosphorylates CIT-K. ( a ) Schematic diagram of CIT-K structure illustrating the phosphorylated sites identified by MS. The GST- tagged fragments used for the in vitro phosphorylation assays shown in ( b ), ( c ) and ( d ) are depicted at the bottom. ( b ) GST-tagged CIT-K polypeptides, GST alone and the positive control MBP were incubated with (+) or without (−) recombinant Aurora B in the presence of [γ- 32 P] ATP. The reactions were then separated by SDS-PAGE, transferred onto nitrocellulose membranes and exposed at −80°C. The Ponceau S staining of the protein loading is shown at the bottom. Aurora B auto-phosphorylation is marked by an asterisk. The numbers on the left indicate the sizes in kilodaltons of the molecular mass marker. ( c ) GST-tagged wild-type CIT-K-CC1 (WT) and S699A mutant polypeptides, GST alone and the positive control MBP (myelin basic protein) were incubated with (+) or without (−) recombinant Aurora B in the presence of [γ- 32 P] ATP. The reactions were then separated by SDS-PAGE, transferred onto nitrocellulose membranes and exposed at −80°C. The protein loading is shown at the bottom. Aurora B auto-phosphorylation is marked by an asterisk. The numbers on the left indicate the sizes in kilodaltons of the molecular mass marker. ( d ) The GST-tagged wild-type CIT-K-C1+PH peptide (WT), along with the S1385A-S1386A-T1387A (TripleA) and S1474A mutant polypeptides, GST alone and the positive control MBP (myelin basic protein) were incubated with (+) or without (−) recombinant Aurora B in the presence of [γ- 32 P] ATP. The reactions were then separated by SDS-PAGE, transferred onto nitrocellulose membranes and exposed at −80°C. The protein loading is shown at the bottom. Aurora B auto-phosphorylation is marked by an asterisk. The numbers on the left indicate the sizes in kilodaltons of the molecular mass marker. ( e ) HeLa Kyoto cells stably expressing Flag::CIT-K or Flag::CIT-K-S699A were treated with an siRNA directed against the CIT-K 3′-UTR for 48 h, blocked in metaphase by thymidine/nocodazole block, released for 90 min and then treated with 10 µM RO3306 for further 15 min. Proteins were extracted and used in a pull-down assay with anti-Flag antibodies. The extracts and pull downs were analysed by western blot to detect KIF14, KIF23, Aurora B and Flag::CIT-K. The numbers on the left indicate the sizes in kilodaltons of the molecular mass marker. ( f ) HeLa Kyoto cells stably expressing Flag::CIT-K or Flag::CIT-K-S699A were treated with siRNAs directed against either a random sequence (control) or 3′-UTR CIT-K for 48 h. During RNAi incubation, cells were synchronized using 2 mM thymidine for 19 h, released for 5 h, treated with 10 µM RO3306 for 13 h, released for 2 h, fixed and stained to detect Flag (red), tubulin (green) and DNA (blue). All images are maximum intensity projections of the three most central z sections; z step = 0.25 µm. Scale bars, 10 µm. ( g ) Quantification of CIT-K midzone localization from the experiments showed in ( f ). No less than 50 early–mid-telophase cells were counted in each experiment, n = 4. Bars indicate standard errors.
Figure Legend Snippet: Aurora B phosphorylates CIT-K. ( a ) Schematic diagram of CIT-K structure illustrating the phosphorylated sites identified by MS. The GST- tagged fragments used for the in vitro phosphorylation assays shown in ( b ), ( c ) and ( d ) are depicted at the bottom. ( b ) GST-tagged CIT-K polypeptides, GST alone and the positive control MBP were incubated with (+) or without (−) recombinant Aurora B in the presence of [γ- 32 P] ATP. The reactions were then separated by SDS-PAGE, transferred onto nitrocellulose membranes and exposed at −80°C. The Ponceau S staining of the protein loading is shown at the bottom. Aurora B auto-phosphorylation is marked by an asterisk. The numbers on the left indicate the sizes in kilodaltons of the molecular mass marker. ( c ) GST-tagged wild-type CIT-K-CC1 (WT) and S699A mutant polypeptides, GST alone and the positive control MBP (myelin basic protein) were incubated with (+) or without (−) recombinant Aurora B in the presence of [γ- 32 P] ATP. The reactions were then separated by SDS-PAGE, transferred onto nitrocellulose membranes and exposed at −80°C. The protein loading is shown at the bottom. Aurora B auto-phosphorylation is marked by an asterisk. The numbers on the left indicate the sizes in kilodaltons of the molecular mass marker. ( d ) The GST-tagged wild-type CIT-K-C1+PH peptide (WT), along with the S1385A-S1386A-T1387A (TripleA) and S1474A mutant polypeptides, GST alone and the positive control MBP (myelin basic protein) were incubated with (+) or without (−) recombinant Aurora B in the presence of [γ- 32 P] ATP. The reactions were then separated by SDS-PAGE, transferred onto nitrocellulose membranes and exposed at −80°C. The protein loading is shown at the bottom. Aurora B auto-phosphorylation is marked by an asterisk. The numbers on the left indicate the sizes in kilodaltons of the molecular mass marker. ( e ) HeLa Kyoto cells stably expressing Flag::CIT-K or Flag::CIT-K-S699A were treated with an siRNA directed against the CIT-K 3′-UTR for 48 h, blocked in metaphase by thymidine/nocodazole block, released for 90 min and then treated with 10 µM RO3306 for further 15 min. Proteins were extracted and used in a pull-down assay with anti-Flag antibodies. The extracts and pull downs were analysed by western blot to detect KIF14, KIF23, Aurora B and Flag::CIT-K. The numbers on the left indicate the sizes in kilodaltons of the molecular mass marker. ( f ) HeLa Kyoto cells stably expressing Flag::CIT-K or Flag::CIT-K-S699A were treated with siRNAs directed against either a random sequence (control) or 3′-UTR CIT-K for 48 h. During RNAi incubation, cells were synchronized using 2 mM thymidine for 19 h, released for 5 h, treated with 10 µM RO3306 for 13 h, released for 2 h, fixed and stained to detect Flag (red), tubulin (green) and DNA (blue). All images are maximum intensity projections of the three most central z sections; z step = 0.25 µm. Scale bars, 10 µm. ( g ) Quantification of CIT-K midzone localization from the experiments showed in ( f ). No less than 50 early–mid-telophase cells were counted in each experiment, n = 4. Bars indicate standard errors.

Techniques Used: Mass Spectrometry, In Vitro, Positive Control, Incubation, Recombinant, SDS Page, Staining, Marker, Mutagenesis, Stable Transfection, Expressing, Blocking Assay, Pull Down Assay, Western Blot, Sequencing

CIT-K phosphorylates INCENP. ( a ) Schematic diagram of INCENP structure illustrating the phosphorylated sites identified by MS. The GST-tagged fragments used for the in vitro phosphorylation assays shown in ( b ), ( c ) and ( d ) are depicted at the bottom. ( b ) GST-tagged INCENP polypeptides, GST alone, and the positive control MBP (myelin basic protein) were incubated with GST-tagged CIT-K kinase domain or KD-CIT-K kinase domain in the presence of [γ- 32 P] ATP. The reactions were then separated by SDS-PAGE, transferred onto nitrocellulose membranes, and exposed at −80°C. The Ponceau S staining of the protein loading is shown at the bottom. The asterisks mark the molecular positioning of the respective proteins. The dagger (†) indicates CIT-K auto-phosphorylation. The numbers on the left indicate the sizes in kilodaltons of the molecular mass marker. ( c ) GST alone, GST-tagged INCENP 783–918 and GST-tagged INCENP mutants (T844A, TSS/AAA and T844A + TSS/AAA), were incubated with GST-tagged CIT-K kinase domain or KD-CIT-K kinase domain in the presence of [γ- 32 P] ATP. The reactions were then separated by SDS-PAGE, transferred onto nitrocellulose membranes, and exposed at −80°C. The protein loading is shown at the bottom. An asterisk marks CIT-K auto-phosphorylation. The numbers on the left indicate the sizes in kilodaltons of the molecular mass marker. ( d ) GST alone and GST-tagged INCENP 783–918 were incubated in the presence or absence of GST-tagged CIT-K kinase domain or KD-CIT-K kinase domain, using non-radioactive ATP. The reactions were then separated by SDS-PAGE and analysed by western blot to detect phosphorylated INCENP. The protein loading is shown at the bottom. The numbers on the left indicate the sizes in kilodaltons of the molecular mass marker. ( e ) HeLa Kyoto cells were treated with siRNAs directed against either a random sequence (control) or CIT-K for 48 h. During RNAi incubation, cells were synchronized using 2 mM thymidine for 19 h, released for 5 h, treated with 10 µM RO3306 for 13 h, released for 2 h, fixed and stained to detect phosphorylated INCENP (green), tubulin (red) and DNA (blue). Insets show 2× magnification of the midbody. The box plot showing the quantification of pTSS fluorescence levels at the midbody is shown on the right. The intensity of pTSS INCENP fluorescence at the midbody was calculated using the formula shown, where the mean fluorescence intensity was measured at the midbody ( I M ) and the mean background fluorescence intensity was measured within an identical area inside the cytoplasm ( I C ). The numbers of cells counted are detailed below each plot. Scale bars, 10 µm. ** p
Figure Legend Snippet: CIT-K phosphorylates INCENP. ( a ) Schematic diagram of INCENP structure illustrating the phosphorylated sites identified by MS. The GST-tagged fragments used for the in vitro phosphorylation assays shown in ( b ), ( c ) and ( d ) are depicted at the bottom. ( b ) GST-tagged INCENP polypeptides, GST alone, and the positive control MBP (myelin basic protein) were incubated with GST-tagged CIT-K kinase domain or KD-CIT-K kinase domain in the presence of [γ- 32 P] ATP. The reactions were then separated by SDS-PAGE, transferred onto nitrocellulose membranes, and exposed at −80°C. The Ponceau S staining of the protein loading is shown at the bottom. The asterisks mark the molecular positioning of the respective proteins. The dagger (†) indicates CIT-K auto-phosphorylation. The numbers on the left indicate the sizes in kilodaltons of the molecular mass marker. ( c ) GST alone, GST-tagged INCENP 783–918 and GST-tagged INCENP mutants (T844A, TSS/AAA and T844A + TSS/AAA), were incubated with GST-tagged CIT-K kinase domain or KD-CIT-K kinase domain in the presence of [γ- 32 P] ATP. The reactions were then separated by SDS-PAGE, transferred onto nitrocellulose membranes, and exposed at −80°C. The protein loading is shown at the bottom. An asterisk marks CIT-K auto-phosphorylation. The numbers on the left indicate the sizes in kilodaltons of the molecular mass marker. ( d ) GST alone and GST-tagged INCENP 783–918 were incubated in the presence or absence of GST-tagged CIT-K kinase domain or KD-CIT-K kinase domain, using non-radioactive ATP. The reactions were then separated by SDS-PAGE and analysed by western blot to detect phosphorylated INCENP. The protein loading is shown at the bottom. The numbers on the left indicate the sizes in kilodaltons of the molecular mass marker. ( e ) HeLa Kyoto cells were treated with siRNAs directed against either a random sequence (control) or CIT-K for 48 h. During RNAi incubation, cells were synchronized using 2 mM thymidine for 19 h, released for 5 h, treated with 10 µM RO3306 for 13 h, released for 2 h, fixed and stained to detect phosphorylated INCENP (green), tubulin (red) and DNA (blue). Insets show 2× magnification of the midbody. The box plot showing the quantification of pTSS fluorescence levels at the midbody is shown on the right. The intensity of pTSS INCENP fluorescence at the midbody was calculated using the formula shown, where the mean fluorescence intensity was measured at the midbody ( I M ) and the mean background fluorescence intensity was measured within an identical area inside the cytoplasm ( I C ). The numbers of cells counted are detailed below each plot. Scale bars, 10 µm. ** p

Techniques Used: Mass Spectrometry, In Vitro, Positive Control, Incubation, SDS Page, Staining, Marker, Western Blot, Sequencing, Fluorescence

59) Product Images from "Selective inhibition of the p38 alternative activation pathway in infiltrating T cells inhibits pancreatic cancer progression"

Article Title: Selective inhibition of the p38 alternative activation pathway in infiltrating T cells inhibits pancreatic cancer progression

Journal: Nature medicine

doi: 10.1038/nm.3957

Gadd45α residues 71–85 bind p38 and inhibits T cell proliferation and effector function ( a, b ) Recombinant soluble p38 was incubated with beads coated with full length Gadd45α (G45α FL) and the indicated fragments in PBS at 37°C for 1 hr. Eluates were immunoblotted for p38 and GST. Results are representative of two independent experiments. ( c ) Recombinant p38 was activated with Zap70, MKK6, or IVK buffer alone at 30°C for 1 hr, then incubated with the indicated GST-Gadd45α fusion proteins at 30°C for 20 min. ATF2 was added with 10 μCi [γ- 32 P] ATP for an additional 30 min. 32 P incorporation was detected by SDS-PAGE and autoradiography. Results are representative of two independent experiments. ( d ) Recombinant p38 was phosphorylated by Zap70 or IVK buffer alone and then incubated with (11R) 71–85 or (11R) Scr at 30°C for 20 min. ATF2 was added with 10 μCi [γ- 32 P] ATP for an additional 30 min, and 32 P incorporation was assessed. Results are representative of two independent experiments. ( e ) Purified B6 splenic T cells were incubated or not with FITC-labeled (11R) 71–85 or (11R) Scr for 2 hr. Cells were washed and analyzed at the indicated times for retention of the fluorescently-labeled peptides. Results are representative of two independent experiments. ( f ) Purified T cells were incubated as in e , then stimulated with plate-bound anti-CD3/CD28 or PMA/ionomycin for 1 hr, lysed, and immunoblotted for phospho-p38 (P-p38). Results are representative of three independent experiments. ( g ) Purified T or B cells were incubated in e and stimulated with plate-bound anti-CD3/CD28 (T cells) or anti-IgM-Fab 2 ′ (B cells) for 48 hr. [ 3 H]-thymidine was added 16 hr before cells were harvested. Results are representative of three independent experiments. ( h ) Freshly purified T cells were incubated as in e , then stimulated with the indicated amounts of plate-bound anti-CD3 and a fixed amount of plate-bound anti-CD28 or the indicated concentrations of PMA and a fixed concentration of ionomycin for 48 hr. [ 3 H]-thymidine was added 16 hr before cells were harvested. Results are representative of three independent experiments. ( i ) Purified T cells were incubated or not with (11R) 71–85 or (11R) Scr as in e, then stimulated with anti-CD3/CD28 or PMA and ionomycin for 48 hr before collecting the supernatants and measuring TNF-α by ELISA. Results are representative of three independent experiments.
Figure Legend Snippet: Gadd45α residues 71–85 bind p38 and inhibits T cell proliferation and effector function ( a, b ) Recombinant soluble p38 was incubated with beads coated with full length Gadd45α (G45α FL) and the indicated fragments in PBS at 37°C for 1 hr. Eluates were immunoblotted for p38 and GST. Results are representative of two independent experiments. ( c ) Recombinant p38 was activated with Zap70, MKK6, or IVK buffer alone at 30°C for 1 hr, then incubated with the indicated GST-Gadd45α fusion proteins at 30°C for 20 min. ATF2 was added with 10 μCi [γ- 32 P] ATP for an additional 30 min. 32 P incorporation was detected by SDS-PAGE and autoradiography. Results are representative of two independent experiments. ( d ) Recombinant p38 was phosphorylated by Zap70 or IVK buffer alone and then incubated with (11R) 71–85 or (11R) Scr at 30°C for 20 min. ATF2 was added with 10 μCi [γ- 32 P] ATP for an additional 30 min, and 32 P incorporation was assessed. Results are representative of two independent experiments. ( e ) Purified B6 splenic T cells were incubated or not with FITC-labeled (11R) 71–85 or (11R) Scr for 2 hr. Cells were washed and analyzed at the indicated times for retention of the fluorescently-labeled peptides. Results are representative of two independent experiments. ( f ) Purified T cells were incubated as in e , then stimulated with plate-bound anti-CD3/CD28 or PMA/ionomycin for 1 hr, lysed, and immunoblotted for phospho-p38 (P-p38). Results are representative of three independent experiments. ( g ) Purified T or B cells were incubated in e and stimulated with plate-bound anti-CD3/CD28 (T cells) or anti-IgM-Fab 2 ′ (B cells) for 48 hr. [ 3 H]-thymidine was added 16 hr before cells were harvested. Results are representative of three independent experiments. ( h ) Freshly purified T cells were incubated as in e , then stimulated with the indicated amounts of plate-bound anti-CD3 and a fixed amount of plate-bound anti-CD28 or the indicated concentrations of PMA and a fixed concentration of ionomycin for 48 hr. [ 3 H]-thymidine was added 16 hr before cells were harvested. Results are representative of three independent experiments. ( i ) Purified T cells were incubated or not with (11R) 71–85 or (11R) Scr as in e, then stimulated with anti-CD3/CD28 or PMA and ionomycin for 48 hr before collecting the supernatants and measuring TNF-α by ELISA. Results are representative of three independent experiments.

Techniques Used: Recombinant, Incubation, SDS Page, Autoradiography, Purification, Labeling, Concentration Assay, Enzyme-linked Immunosorbent Assay

60) Product Images from "A novel in vitro pancreatic carcinogenesis model"

Article Title: A novel in vitro pancreatic carcinogenesis model

Journal: Toxicology letters

doi: 10.1016/j.toxlet.2011.01.012

BaP-DNA adduct accumulation in pancreatic ductal cells. (A) HPDE6-C7 cells were transfected with control, AhR, and BRCA1-specific siRNA for 72 hr and further incubated with 2.5 or 5 nM [ 3 H]BaP for 24 hr. Then DNA incorporated [ 3 H]BaP were measured from purified genomic DNA using a liquid scintillation counter. (B) Efficiency of knockdown was measured by standard Western blotting. (C) HPDE6-C7 cells were transfected with control, AhR, and BRCA1-specific siRNA for 72 hr and further incubated with 5 μM BaP for 24 h. Then post-labeled with [γ- 32 P]ATP and [ 32 P] labeled DNA adduct was detected as described in material and methods. 1 μM of BPDE treated HPDE6-C7 cells were used as positive control for DNA-BPDE adduct spot. ***, p
Figure Legend Snippet: BaP-DNA adduct accumulation in pancreatic ductal cells. (A) HPDE6-C7 cells were transfected with control, AhR, and BRCA1-specific siRNA for 72 hr and further incubated with 2.5 or 5 nM [ 3 H]BaP for 24 hr. Then DNA incorporated [ 3 H]BaP were measured from purified genomic DNA using a liquid scintillation counter. (B) Efficiency of knockdown was measured by standard Western blotting. (C) HPDE6-C7 cells were transfected with control, AhR, and BRCA1-specific siRNA for 72 hr and further incubated with 5 μM BaP for 24 h. Then post-labeled with [γ- 32 P]ATP and [ 32 P] labeled DNA adduct was detected as described in material and methods. 1 μM of BPDE treated HPDE6-C7 cells were used as positive control for DNA-BPDE adduct spot. ***, p

Techniques Used: Transfection, Incubation, Purification, Western Blot, Labeling, Positive Control

61) 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

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

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

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.24.6.2423-2443.2004

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

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

63) Product Images from "α-Catenin phosphorylation promotes intercellular adhesion through a dual-kinase mechanism"

Article Title: α-Catenin phosphorylation promotes intercellular adhesion through a dual-kinase mechanism

Journal: Journal of Cell Science

doi: 10.1242/jcs.163824

Identification of major CK2 and CK1 sites in α-cat. (A) Identification of S641 as the major CK2 site. Autoradiograph of [γ- 32 P]ATP in vitro kinase labeling of recombinant full-length (FL) and S641A (A, alanine) αE-cat. The timecourse
Figure Legend Snippet: Identification of major CK2 and CK1 sites in α-cat. (A) Identification of S641 as the major CK2 site. Autoradiograph of [γ- 32 P]ATP in vitro kinase labeling of recombinant full-length (FL) and S641A (A, alanine) αE-cat. The timecourse

Techniques Used: Autoradiography, In Vitro, Labeling, Recombinant

64) Product Images from "The Nucleophosmin-Anaplastic Lymphoma Kinase Oncogene Interacts, Activates, and Uses the Kinase PIKfyve to Increase Invasiveness *"

Article Title: The Nucleophosmin-Anaplastic Lymphoma Kinase Oncogene Interacts, Activates, and Uses the Kinase PIKfyve to Increase Invasiveness *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M111.227512

The PI 5-kinase PIKfyve interacts with NPM-ALK. NPM-ALK was immunoprecipitated ( IP ) from NIH-3T3 NPM-ALK(+) cells, and immunoprecipitates were submitted to a lipid kinase assay with PtdIns vesicles (200 μ m ) and [γ- 32 P]ATP in the presence
Figure Legend Snippet: The PI 5-kinase PIKfyve interacts with NPM-ALK. NPM-ALK was immunoprecipitated ( IP ) from NIH-3T3 NPM-ALK(+) cells, and immunoprecipitates were submitted to a lipid kinase assay with PtdIns vesicles (200 μ m ) and [γ- 32 P]ATP in the presence

Techniques Used: Immunoprecipitation, Kinase Assay

65) Product Images from "GCN2 phosphorylates HIV-1 integrase and decreases HIV-1 replication by limiting viral integration"

Article Title: GCN2 phosphorylates HIV-1 integrase and decreases HIV-1 replication by limiting viral integration

Journal: Scientific Reports

doi: 10.1038/s41598-017-02276-0

GCN2 in vitro phosphorylation assay. ( a ) Phosphorylation of HIV-1 IN catalyzed by GCN2. Lane 1, HIV-1 IN (2000 nM) alone. Lane 2 to 7, GCN2 (24 nM) was incubated with [γ- 32 P]-ATP and increasing concentrations of HIV-1 IN (0, 100, 225, 450, 1000, 2000 nM). ( b ) Competition assay. GCN2 phosphorylation of increasing concentrations of eIF2α (from 100 nM to 800 nM following a 2-fold increment) with radiolabeled [γ- 32 P]-ATP, in the presence or not of HIV-1 IN (2 µM). ( c ) Impact of HIV-1 IN oligomerization on phosphorylation by GCN2. Lane 1, no IN. Lanes 2 to 6, HIV-1 IN WT or mutants (700 nM).
Figure Legend Snippet: GCN2 in vitro phosphorylation assay. ( a ) Phosphorylation of HIV-1 IN catalyzed by GCN2. Lane 1, HIV-1 IN (2000 nM) alone. Lane 2 to 7, GCN2 (24 nM) was incubated with [γ- 32 P]-ATP and increasing concentrations of HIV-1 IN (0, 100, 225, 450, 1000, 2000 nM). ( b ) Competition assay. GCN2 phosphorylation of increasing concentrations of eIF2α (from 100 nM to 800 nM following a 2-fold increment) with radiolabeled [γ- 32 P]-ATP, in the presence or not of HIV-1 IN (2 µM). ( c ) Impact of HIV-1 IN oligomerization on phosphorylation by GCN2. Lane 1, no IN. Lanes 2 to 6, HIV-1 IN WT or mutants (700 nM).

Techniques Used: In Vitro, Phosphorylation Assay, Incubation, Competitive Binding Assay

66) Product Images from "Insights into the regulation of eukaryotic elongation factor 2 kinase and the interplay between its domains"

Article Title: Insights into the regulation of eukaryotic elongation factor 2 kinase and the interplay between its domains

Journal: Biochemical Journal

doi: 10.1042/BJ20111536

Regulation of the binding of eEF2K to ATP studied by STD NMR ( A ) Binding of ATP-γS to eEF2K as a function of Ca 2+ /CaM. The same section of the 1 H spectrum of a 1 mM ATP-γS solution is shown under four experimental conditions: (i) 1 H NMR spectrum of ATP-γS; (ii) 1 H STD spectrum of ATP-γS showing that the STD pulses do not saturate ATP-γS; (iii) same 1 H STD spectrum of ATP-γS in the presence of 9 μM eEF2K[48–336] and eEF2K[490–725]; and (iv) in the presence of a mixture of 9 μM eEF2K[48–336] and eEF2K[490–725] and 1 mM CaCl 2 and 100 μM CaM respectively. Binding of ATP-γS to Ca 2+ /CaM could be excluded because of the absence of STD signals in an ATP-γS Ca 2+ /CaM solution (results not shown). The inset shows the structure of ATP-γS. ( B ) Dependence of activity and ATP-cross-linking on Ca 2+ /CaM. To test ATP binding, recombinant GST–eEF2K was incubated with [α- 32 P]ATP in the absence or presence of Ca 2+ ions or CaM, as indicated. Samples were subjected to UV irradiation as described in the Experimental section. In parallel, other samples were incubated with [γ- 32 P]ATP and eEF2 to examine autophosphorylation of eEF2K and activity against eEF2. Equal levels of eEF2K protein were confirmed by staining the gel with Coomassie Brilliant Blue (bottom panel). The other three panels show autoradiographs of SDS/PAGE.
Figure Legend Snippet: Regulation of the binding of eEF2K to ATP studied by STD NMR ( A ) Binding of ATP-γS to eEF2K as a function of Ca 2+ /CaM. The same section of the 1 H spectrum of a 1 mM ATP-γS solution is shown under four experimental conditions: (i) 1 H NMR spectrum of ATP-γS; (ii) 1 H STD spectrum of ATP-γS showing that the STD pulses do not saturate ATP-γS; (iii) same 1 H STD spectrum of ATP-γS in the presence of 9 μM eEF2K[48–336] and eEF2K[490–725]; and (iv) in the presence of a mixture of 9 μM eEF2K[48–336] and eEF2K[490–725] and 1 mM CaCl 2 and 100 μM CaM respectively. Binding of ATP-γS to Ca 2+ /CaM could be excluded because of the absence of STD signals in an ATP-γS Ca 2+ /CaM solution (results not shown). The inset shows the structure of ATP-γS. ( B ) Dependence of activity and ATP-cross-linking on Ca 2+ /CaM. To test ATP binding, recombinant GST–eEF2K was incubated with [α- 32 P]ATP in the absence or presence of Ca 2+ ions or CaM, as indicated. Samples were subjected to UV irradiation as described in the Experimental section. In parallel, other samples were incubated with [γ- 32 P]ATP and eEF2 to examine autophosphorylation of eEF2K and activity against eEF2. Equal levels of eEF2K protein were confirmed by staining the gel with Coomassie Brilliant Blue (bottom panel). The other three panels show autoradiographs of SDS/PAGE.

Techniques Used: Binding Assay, Nuclear Magnetic Resonance, Chick Chorioallantoic Membrane Assay, Activity Assay, Recombinant, Incubation, Irradiation, Staining, SDS Page

Activities of a range of fragments of eEF2K ( A ) The indicated fragments, all GST fusions containing the catalytic domain of eEF2K, were incubated with [γ- 32 P]ATP in the presence of Ca 2+ /CaM and, where indicated, the eEF2K[478–725] fragment. The Figure is an autoradiograph of the stained gel. The positions of the catalytic domain fragments (CD) and eEF2K[478–725] are shown. ( B ) As ( A ), using the indicated GST–eEF2K fragments alone, or in the absence or presence of Ca 2+ /CaM. The faster-migrating radiolabelled species represent fragments created by proteolytic degradation. ( C ) GST–eEF2K[64–356] and eEF2K[478–725] fragments were incubated with the MH-1 peptide in the presence of [γ- 32 P]ATP with or without Ca 2+ ions and CaM for the indicated times.
Figure Legend Snippet: Activities of a range of fragments of eEF2K ( A ) The indicated fragments, all GST fusions containing the catalytic domain of eEF2K, were incubated with [γ- 32 P]ATP in the presence of Ca 2+ /CaM and, where indicated, the eEF2K[478–725] fragment. The Figure is an autoradiograph of the stained gel. The positions of the catalytic domain fragments (CD) and eEF2K[478–725] are shown. ( B ) As ( A ), using the indicated GST–eEF2K fragments alone, or in the absence or presence of Ca 2+ /CaM. The faster-migrating radiolabelled species represent fragments created by proteolytic degradation. ( C ) GST–eEF2K[64–356] and eEF2K[478–725] fragments were incubated with the MH-1 peptide in the presence of [γ- 32 P]ATP with or without Ca 2+ ions and CaM for the indicated times.

Techniques Used: Incubation, Chick Chorioallantoic Membrane Assay, Autoradiography, Staining

Activities of catalytic fragments against eEF2 or the MH-1 peptide depends on the presence of the eEF2K[478–725] fragment ( A ) GST–eEF2K[1–402] was incubated with eEF2 in the presence of [γ- 32 P]ATP in the absence or presence of Ca 2+ ions, CaM and/or the eEF2K[478–725] fragment as indicated. The Figure shows an autoradiograph of the resulting gels. ( B – D ) GST–eEF2K[1–402], [76–356] or [76–402] was incubated with the MH-1 peptide in the presence of [γ- 32 P]ATP in the presence of Ca 2+ ions and CaM with or without the eEF2K[478–725] fragment. ( E ) The indicated proteins were incubated with eEF2 and non-radioactive ATP. Reaction products were analysed by SDS/PAGE and Western blotting with the anti-eEF2 (P)Thr 56 antibody (pT56) or anti-eEF2 as a loading control.
Figure Legend Snippet: Activities of catalytic fragments against eEF2 or the MH-1 peptide depends on the presence of the eEF2K[478–725] fragment ( A ) GST–eEF2K[1–402] was incubated with eEF2 in the presence of [γ- 32 P]ATP in the absence or presence of Ca 2+ ions, CaM and/or the eEF2K[478–725] fragment as indicated. The Figure shows an autoradiograph of the resulting gels. ( B – D ) GST–eEF2K[1–402], [76–356] or [76–402] was incubated with the MH-1 peptide in the presence of [γ- 32 P]ATP in the presence of Ca 2+ ions and CaM with or without the eEF2K[478–725] fragment. ( E ) The indicated proteins were incubated with eEF2 and non-radioactive ATP. Reaction products were analysed by SDS/PAGE and Western blotting with the anti-eEF2 (P)Thr 56 antibody (pT56) or anti-eEF2 as a loading control.

Techniques Used: Incubation, Chick Chorioallantoic Membrane Assay, Autoradiography, SDS Page, Western Blot

67) Product Images from "The chromosomal passenger complex controls the function of endosomal sorting complex required for transport-III Snf7 proteins during cytokinesis"

Article Title: The chromosomal passenger complex controls the function of endosomal sorting complex required for transport-III Snf7 proteins during cytokinesis

Journal: Open Biology

doi: 10.1098/rsob.120070

Aurora B phosphorylates CHMP4C in vitro and in vivo . ( a ) GST-tagged CHMP4 proteins, GST alone or the positive control MBP (myelin basic protein) were incubated with (+) or without (−) recombinant Aurora B in the presence of [γ- 32 P] ATP. The reactions were then separated by SDS-PAGE, and gels stained with Coomassie Blue, dried and exposed at −80°C. The Coomassie Blue staining of the protein loading is shown at the bottom. Note that Aurora B is auto-phosphorylated and co-migrated with GST::CHMP4B. The numbers on the right indicate the sizes (kDa) of the molecular mass marker. ( b ) GST-tagged full-length CHMP4C (FL), GST-tagged N-terminal CHMP4Cα12, GST-tagged C-terminal CHMP4Cα345, GST alone and the positive control MBP were incubated with (+) or without (−) recombinant Aurora B in the presence of [γ- 32 P] ATP. Products of the reactions were then separated by SDS-PAGE and the gels stained with Coomassie Blue, dried and exposed at −80°C. The Coomassie Blue staining of the protein loading is shown at the bottom. The numbers on the right indicate the sizes (kDa) of the molecular mass marker. ( c ) GST-tagged wild-type CHMP4Cα345 (WT), the two GST:: CHMP4Cα345 variants containing S to A mutations at position 210 (S210A) or at position 210, 214 and 215 (StripleA), GST alone and the positive control MBP were incubated with (+) or without (−) recombinant Aurora B in the presence of [γ- 32 P] ATP. Products of the reactions were then separated by SDS-PAGE and the gels stained with Coomassie Blue, dried and exposed at −80°C. The Coomassie Blue staining of the protein loading is shown at the bottom. The numbers on the right indicate the sizes (kDa) of the molecular mass marker. ( d ) HeLa cells were transfected with GFP::CHMP4C, synchronized in metaphase with thymidine/nocodazole block and then released into medium containing either ZM447439 or its solvent DMSO as control. Proteins were extracted, separated by SDS-PAGE, transferred onto PVDF membranes and analysed by Western blot to detect the variant of CHMP4C phosphorylated at serine 210, 214 and 215 (phospho-CHMP4C), cyclin B, Borealin, GFP::CHMP4C and tubulin as loading control. The numbers indicate the sizes (kDa) of the molecular mass marker.
Figure Legend Snippet: Aurora B phosphorylates CHMP4C in vitro and in vivo . ( a ) GST-tagged CHMP4 proteins, GST alone or the positive control MBP (myelin basic protein) were incubated with (+) or without (−) recombinant Aurora B in the presence of [γ- 32 P] ATP. The reactions were then separated by SDS-PAGE, and gels stained with Coomassie Blue, dried and exposed at −80°C. The Coomassie Blue staining of the protein loading is shown at the bottom. Note that Aurora B is auto-phosphorylated and co-migrated with GST::CHMP4B. The numbers on the right indicate the sizes (kDa) of the molecular mass marker. ( b ) GST-tagged full-length CHMP4C (FL), GST-tagged N-terminal CHMP4Cα12, GST-tagged C-terminal CHMP4Cα345, GST alone and the positive control MBP were incubated with (+) or without (−) recombinant Aurora B in the presence of [γ- 32 P] ATP. Products of the reactions were then separated by SDS-PAGE and the gels stained with Coomassie Blue, dried and exposed at −80°C. The Coomassie Blue staining of the protein loading is shown at the bottom. The numbers on the right indicate the sizes (kDa) of the molecular mass marker. ( c ) GST-tagged wild-type CHMP4Cα345 (WT), the two GST:: CHMP4Cα345 variants containing S to A mutations at position 210 (S210A) or at position 210, 214 and 215 (StripleA), GST alone and the positive control MBP were incubated with (+) or without (−) recombinant Aurora B in the presence of [γ- 32 P] ATP. Products of the reactions were then separated by SDS-PAGE and the gels stained with Coomassie Blue, dried and exposed at −80°C. The Coomassie Blue staining of the protein loading is shown at the bottom. The numbers on the right indicate the sizes (kDa) of the molecular mass marker. ( d ) HeLa cells were transfected with GFP::CHMP4C, synchronized in metaphase with thymidine/nocodazole block and then released into medium containing either ZM447439 or its solvent DMSO as control. Proteins were extracted, separated by SDS-PAGE, transferred onto PVDF membranes and analysed by Western blot to detect the variant of CHMP4C phosphorylated at serine 210, 214 and 215 (phospho-CHMP4C), cyclin B, Borealin, GFP::CHMP4C and tubulin as loading control. The numbers indicate the sizes (kDa) of the molecular mass marker.

Techniques Used: In Vitro, In Vivo, Positive Control, Incubation, Recombinant, SDS Page, Staining, Marker, Transfection, Blocking Assay, Western Blot, Variant Assay

68) Product Images from "Stem Cell Factor-Inducible MITF-M Expression in Therapeutics for Acquired Skin Hyperpigmentation"

Article Title: Stem Cell Factor-Inducible MITF-M Expression in Therapeutics for Acquired Skin Hyperpigmentation

Journal: Theranostics

doi: 10.7150/thno.39066

KIT-catalyzed tyrosine kinase activity in cell-free condition. Catalytically active rhKIT was reacted with poly(Glu,Tyr 4:1) peptide as an exogenous substrate in the presence of BPT. (A) In vitro kinase activity as the count per minute (cpm) by incorporating [ 32 P] from [γ- 32 P]ATP to the peptide substrate. Data are mean ± SEM. * P
Figure Legend Snippet: KIT-catalyzed tyrosine kinase activity in cell-free condition. Catalytically active rhKIT was reacted with poly(Glu,Tyr 4:1) peptide as an exogenous substrate in the presence of BPT. (A) In vitro kinase activity as the count per minute (cpm) by incorporating [ 32 P] from [γ- 32 P]ATP to the peptide substrate. Data are mean ± SEM. * P

Techniques Used: Activity Assay, In Vitro

69) 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

70) Product Images from "Protein Phosphatase-1? Interacts with and Dephosphorylates Polycystin-1"

Article Title: Protein Phosphatase-1? Interacts with and Dephosphorylates Polycystin-1

Journal: PLoS ONE

doi: 10.1371/journal.pone.0036798

Dephosphorylation of PC1 by PP1α. To determine whether PP1α can dephosphorylate PKA-phosphorylated PC1, a GST-PC1 C-tail fusion protein (GST-HT 193 ) was purified from bacteria, bound to GSH-agarose beads, and phosphorylated by PKA with [γ- 32 P]ATP. Unincorporated 32 P was removed by washing the fusion protein with an excess of cold ATP in PP1α or PP2B reaction buffer. The radiolabeled protein was then incubated in the presence or absence of purified, recombinant PP1α or PP2B (calcineurin) for 0–2 h. Aliquots of the reaction were removed and tested for phosphatase activity against the generic substrate p-nitrophenyl phosphate (pNPP) or frozen on dry ice in 1× SDS-PAGE sample buffer to terminate the phosphatase reaction. Terminated reactions were resolved by SDS-PAGE, and phosphorylated fusion protein was detected first by autoradiography (AR) followed by immunoblotting.
Figure Legend Snippet: Dephosphorylation of PC1 by PP1α. To determine whether PP1α can dephosphorylate PKA-phosphorylated PC1, a GST-PC1 C-tail fusion protein (GST-HT 193 ) was purified from bacteria, bound to GSH-agarose beads, and phosphorylated by PKA with [γ- 32 P]ATP. Unincorporated 32 P was removed by washing the fusion protein with an excess of cold ATP in PP1α or PP2B reaction buffer. The radiolabeled protein was then incubated in the presence or absence of purified, recombinant PP1α or PP2B (calcineurin) for 0–2 h. Aliquots of the reaction were removed and tested for phosphatase activity against the generic substrate p-nitrophenyl phosphate (pNPP) or frozen on dry ice in 1× SDS-PAGE sample buffer to terminate the phosphatase reaction. Terminated reactions were resolved by SDS-PAGE, and phosphorylated fusion protein was detected first by autoradiography (AR) followed by immunoblotting.

Techniques Used: De-Phosphorylation Assay, Purification, Incubation, Recombinant, Activity Assay, SDS Page, Autoradiography

71) Product Images from "Cortactin Tyrosine Phosphorylation Requires Rac1 Activity and Association with the Cortical Actin Cytoskeleton"

Article Title: Cortactin Tyrosine Phosphorylation Requires Rac1 Activity and Association with the Cortical Actin Cytoskeleton

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E02-11-0753

Tyrosine phosphorylation of cortactin requires the amino-terminal domain. (A) Diagram of cortactin expression constructs. The cortical targeting domain (N-term) consists of the amino-terminal acidic (NTA) Arp2/3 binding site and the F-actin binding site within the fourth 37 amino acid tandem repeat. The position of the three tyrosine residues phosphorylated by Src and other kinases within the proline-rich carboxyl-terminal domain (C-term) are shown. Numbers represent amino acids encoded by each construct. (B) Differential tyrosine phosphorylation of cortactin tyrosine point mutants in vivo. Individual and serial point mutants (Y-F) representing all possible mutational combinations of the three mapped Src phosphorylation sites were introduced into a cytomegalovirus-driven FLAG-tagged full-length cortactin expression construct and transfected into 5Hd47 fibroblasts. After 18 h, cells were lysed and recombinant cortactin proteins were immunoprecipitated with anti-FLAG agarose beads. Immune complexes were resolved by 8% SDS-PAGE and analyzed for tyrosine phosphorylation by Western blotting with 4G10 (anti-pTyr). The blot was then stripped and reprobed with 4F11 (anti-cortactin) to verify the equal presence of FLAG-cortactin proteins. The codon positions of mutated tyrosine to phenylalanine residues are indicated at the top. FL, full-length, wild-type cortactin. (C) Tyrosine phosphorylation of cortactin domains. 10T1/2 and 5Hd47 cells were transfected with either empty FLAG-vector (vector), full-length wild-type cortactin (FL), full-length 421F/466F/482F cortactin (FL-T), cortactin N-term (NT), cortactin C-term (CT), or cortactin C-term 421F/466F/482F (CT-T) domains. Cells were lysed and FLAG-cortactin proteins were analyzed for phosphotyrosine content as in B (top). Blots were then stripped and the presence of cortactin domains verified by blotting with a mixture of anti-N-term and anti-C-term antibodies (bottom). Note the greater amount of full-length cortactin protein analyzed from the 10T1/2 versus the 5Hd47 line. (D) Src phosphorylation of cortactin domains. FLAG-tagged cortactin domains analyzed in C were immunoprecipitated from transfected 10T1/2 cells and subjected to in vitro kinase assay analysis with [γ- 32 P]ATP in the absence (–) or presence (+) of 1 U of purified Src. Phosphorylated FLAG-cortactin proteins were visualized by autoradiography (top) after 10% SDS-PAGE and transfer to polyvinylidene difluoride membranes. The blot was stripped and the presence of FLAG-cortactin proteins was determined by Western blotting as in C (bottom). The results are indicative of at least three independent experiments.
Figure Legend Snippet: Tyrosine phosphorylation of cortactin requires the amino-terminal domain. (A) Diagram of cortactin expression constructs. The cortical targeting domain (N-term) consists of the amino-terminal acidic (NTA) Arp2/3 binding site and the F-actin binding site within the fourth 37 amino acid tandem repeat. The position of the three tyrosine residues phosphorylated by Src and other kinases within the proline-rich carboxyl-terminal domain (C-term) are shown. Numbers represent amino acids encoded by each construct. (B) Differential tyrosine phosphorylation of cortactin tyrosine point mutants in vivo. Individual and serial point mutants (Y-F) representing all possible mutational combinations of the three mapped Src phosphorylation sites were introduced into a cytomegalovirus-driven FLAG-tagged full-length cortactin expression construct and transfected into 5Hd47 fibroblasts. After 18 h, cells were lysed and recombinant cortactin proteins were immunoprecipitated with anti-FLAG agarose beads. Immune complexes were resolved by 8% SDS-PAGE and analyzed for tyrosine phosphorylation by Western blotting with 4G10 (anti-pTyr). The blot was then stripped and reprobed with 4F11 (anti-cortactin) to verify the equal presence of FLAG-cortactin proteins. The codon positions of mutated tyrosine to phenylalanine residues are indicated at the top. FL, full-length, wild-type cortactin. (C) Tyrosine phosphorylation of cortactin domains. 10T1/2 and 5Hd47 cells were transfected with either empty FLAG-vector (vector), full-length wild-type cortactin (FL), full-length 421F/466F/482F cortactin (FL-T), cortactin N-term (NT), cortactin C-term (CT), or cortactin C-term 421F/466F/482F (CT-T) domains. Cells were lysed and FLAG-cortactin proteins were analyzed for phosphotyrosine content as in B (top). Blots were then stripped and the presence of cortactin domains verified by blotting with a mixture of anti-N-term and anti-C-term antibodies (bottom). Note the greater amount of full-length cortactin protein analyzed from the 10T1/2 versus the 5Hd47 line. (D) Src phosphorylation of cortactin domains. FLAG-tagged cortactin domains analyzed in C were immunoprecipitated from transfected 10T1/2 cells and subjected to in vitro kinase assay analysis with [γ- 32 P]ATP in the absence (–) or presence (+) of 1 U of purified Src. Phosphorylated FLAG-cortactin proteins were visualized by autoradiography (top) after 10% SDS-PAGE and transfer to polyvinylidene difluoride membranes. The blot was stripped and the presence of FLAG-cortactin proteins was determined by Western blotting as in C (bottom). The results are indicative of at least three independent experiments.

Techniques Used: Expressing, Construct, Binding Assay, In Vivo, Transfection, Recombinant, Immunoprecipitation, SDS Page, Western Blot, Plasmid Preparation, In Vitro, Kinase Assay, Purification, Autoradiography

72) Product Images from "The Ca2+-ATPase (SERCA1) Is Inhibited by 4-Aminoquinoline Derivatives through Interference with Catalytic Activation by Ca2+, Whereas the ATPase E2 State Remains Functional *"

Article Title: The Ca2+-ATPase (SERCA1) Is Inhibited by 4-Aminoquinoline Derivatives through Interference with Catalytic Activation by Ca2+, Whereas the ATPase E2 State Remains Functional *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M111.287276

Phosphoenzyme formation by utilization of ATP. The reaction was started by the addition of 10 μ m [γ- 32 P]ATP and 0.95 m m CaCl 2 to protein preincubated with 1 m m EGTA to yield 7 μ m free Ca 2+ . The temperature was 30 °C. See
Figure Legend Snippet: Phosphoenzyme formation by utilization of ATP. The reaction was started by the addition of 10 μ m [γ- 32 P]ATP and 0.95 m m CaCl 2 to protein preincubated with 1 m m EGTA to yield 7 μ m free Ca 2+ . The temperature was 30 °C. See

Techniques Used:

73) Product Images from "Rac1-mediated Mitochondrial H2O2 Generation Regulates MMP-9 Gene Expression in Macrophages via Inhibition of SP-1 and AP-1 *"

Article Title: Rac1-mediated Mitochondrial H2O2 Generation Regulates MMP-9 Gene Expression in Macrophages via Inhibition of SP-1 and AP-1 *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M109.099655

Rac1 null cells have increased SP-1 and AP-1 DNA binding. A , nuclear proteins were isolated from WT and Rac1 null cells, and binding reactions were performed with a consensus SP-1 and AP-1 oligonucleotides labeled with [γ- 32 P]ATP. Cells were transiently
Figure Legend Snippet: Rac1 null cells have increased SP-1 and AP-1 DNA binding. A , nuclear proteins were isolated from WT and Rac1 null cells, and binding reactions were performed with a consensus SP-1 and AP-1 oligonucleotides labeled with [γ- 32 P]ATP. Cells were transiently

Techniques Used: Binding Assay, Isolation, Labeling

74) Product Images from "Protein Kinase C-Mediated Phosphorylation of a Single Serine Residue on the Rat Glial Glutamine Transporter SN1 Governs Its Membrane Trafficking"

Article Title: Protein Kinase C-Mediated Phosphorylation of a Single Serine Residue on the Rat Glial Glutamine Transporter SN1 Governs Its Membrane Trafficking

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.3694-10.2011

PKCα and PKCγ phosphorylate the N-terminal part of SN1 in vitro . Recombinant N-terminal SN1–GST fusion protein bound to glutathione Sepharose underwent phosphorylation in vitro with [γ- 32 P]ATP and PKC-M (catalytic subunit from rat brain) or three recombinant PKC isoforms. After washing of the beads, elution, and SDS-PAGE to purify the fusion protein, the corresponding radioactive bands (localized by phospho-imaging) were cut out of the gel and subjected to thermolysin proteolysis, and phosphopeptides were applied to thin-layer silica sheets and analyzed by horizontal electrophoresis and vertical chromatography (phosphopeptide mapping). A–C , A single phosphopeptide (indicated by arrows) is detected at the same spot during stimulation with PKC-M ( A ), PKCα ( B ), and PKCγ ( C ). D , PKCβ1 does not phosphorylate the N terminal of SN1 (arrow).
Figure Legend Snippet: PKCα and PKCγ phosphorylate the N-terminal part of SN1 in vitro . Recombinant N-terminal SN1–GST fusion protein bound to glutathione Sepharose underwent phosphorylation in vitro with [γ- 32 P]ATP and PKC-M (catalytic subunit from rat brain) or three recombinant PKC isoforms. After washing of the beads, elution, and SDS-PAGE to purify the fusion protein, the corresponding radioactive bands (localized by phospho-imaging) were cut out of the gel and subjected to thermolysin proteolysis, and phosphopeptides were applied to thin-layer silica sheets and analyzed by horizontal electrophoresis and vertical chromatography (phosphopeptide mapping). A–C , A single phosphopeptide (indicated by arrows) is detected at the same spot during stimulation with PKC-M ( A ), PKCα ( B ), and PKCγ ( C ). D , PKCβ1 does not phosphorylate the N terminal of SN1 (arrow).

Techniques Used: In Vitro, Recombinant, SDS Page, Imaging, Electrophoresis, Chromatography

75) Product Images from "Unsaturated Long Chain Free Fatty Acids Are Input Signals of the Salmonella enterica PhoP/PhoQ Regulatory System *"

Article Title: Unsaturated Long Chain Free Fatty Acids Are Input Signals of the Salmonella enterica PhoP/PhoQ Regulatory System *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M113.472829

The autokinase activity of PhoQ is the target of the inhibition by LCUFAs. Increasing concentrations of linoleic acid ( C18:2 ) as indicated ( A ), 0.5 mg ml −1 saturated or unsaturated fatty acids as indicated ( B and C ), or 0.5 mg ml −1 linoleic acid plus the indicated concentration of MgCl 2 ( D ) were added to the Salmonella growth medium. Membranes obtained by cell fractionation, harboring PhoQ or EnvZ, were incubated for 10 min at 37 °C in a reaction medium containing [γ- 32 P]ATP, as described under “Experimental Procedures.” The autophosphorylation reactions were analyzed by SDS-PAGE (12% polyacrylamide) and transferred to nitrocellulose, followed by autoradiography ( top ) or by immunodetection analysis developed with anti-PhoQ Cyt or anti-EnvZ Cyt polyclonal antibodies, respectively ( middle ). The phosphorylation levels and the protein expression levels of PhoQ or EnvZ in each condition assayed were determined by densitometry (as described under “Experimental Procedures”), and the ratio (labeled against immunodetected) was plotted ( bottom ), taking the values obtained with LB alone or with the addition of tergitol or ethanol as 100%, when either saturated or unsaturated fatty acids were used, respectively. Fatty acids are indicated as follows: palmitic acid ( 16:0 ), palmitoleic acid ( 16:1 ), stearic acid ( 18:0 ), oleic acid ( 18:1 ), linoleic acid ( 18:2 ), tergitol ( T ), and ethanol ( E ). Data shown represent results from three independent experiments. Error bars , S.D.
Figure Legend Snippet: The autokinase activity of PhoQ is the target of the inhibition by LCUFAs. Increasing concentrations of linoleic acid ( C18:2 ) as indicated ( A ), 0.5 mg ml −1 saturated or unsaturated fatty acids as indicated ( B and C ), or 0.5 mg ml −1 linoleic acid plus the indicated concentration of MgCl 2 ( D ) were added to the Salmonella growth medium. Membranes obtained by cell fractionation, harboring PhoQ or EnvZ, were incubated for 10 min at 37 °C in a reaction medium containing [γ- 32 P]ATP, as described under “Experimental Procedures.” The autophosphorylation reactions were analyzed by SDS-PAGE (12% polyacrylamide) and transferred to nitrocellulose, followed by autoradiography ( top ) or by immunodetection analysis developed with anti-PhoQ Cyt or anti-EnvZ Cyt polyclonal antibodies, respectively ( middle ). The phosphorylation levels and the protein expression levels of PhoQ or EnvZ in each condition assayed were determined by densitometry (as described under “Experimental Procedures”), and the ratio (labeled against immunodetected) was plotted ( bottom ), taking the values obtained with LB alone or with the addition of tergitol or ethanol as 100%, when either saturated or unsaturated fatty acids were used, respectively. Fatty acids are indicated as follows: palmitic acid ( 16:0 ), palmitoleic acid ( 16:1 ), stearic acid ( 18:0 ), oleic acid ( 18:1 ), linoleic acid ( 18:2 ), tergitol ( T ), and ethanol ( E ). Data shown represent results from three independent experiments. Error bars , S.D.

Techniques Used: Activity Assay, Inhibition, Concentration Assay, Cell Fractionation, Incubation, SDS Page, Autoradiography, Immunodetection, Expressing, Labeling

The inhibition of the autokinase activity of PhoQ by LCUFAs is independent of fadD . 0.5 mg ml −1 linoleic acid ( 18:2 ) was added to the Salmonella wild-type or fadD strains growth medium. Membranes obtained by cell fractionation, harboring PhoQ, were incubated for 10 min at 37 °C in a reaction medium containing [γ- 32 P]ATP, as described under “Experimental Procedures.” The autophosphorylation reactions were analyzed by SDS-PAGE (12% polyacrylamide) and transferred to nitrocellulose, followed by autoradiography ( top ) or immunodetection with anti-PhoQ Cyt polyclonal antibodies ( middle ). The phosphorylation levels and the protein expression levels of PhoQ in each condition assayed were determined by densitometry and the ratio (labeled against immunodetected) was plotted ( bottom ); the value obtained with the addition of ethanol was taken as 100%. 18:2 , linoleic acid; E , ethanol. Data shown represent results from three independent experiments.
Figure Legend Snippet: The inhibition of the autokinase activity of PhoQ by LCUFAs is independent of fadD . 0.5 mg ml −1 linoleic acid ( 18:2 ) was added to the Salmonella wild-type or fadD strains growth medium. Membranes obtained by cell fractionation, harboring PhoQ, were incubated for 10 min at 37 °C in a reaction medium containing [γ- 32 P]ATP, as described under “Experimental Procedures.” The autophosphorylation reactions were analyzed by SDS-PAGE (12% polyacrylamide) and transferred to nitrocellulose, followed by autoradiography ( top ) or immunodetection with anti-PhoQ Cyt polyclonal antibodies ( middle ). The phosphorylation levels and the protein expression levels of PhoQ in each condition assayed were determined by densitometry and the ratio (labeled against immunodetected) was plotted ( bottom ); the value obtained with the addition of ethanol was taken as 100%. 18:2 , linoleic acid; E , ethanol. Data shown represent results from three independent experiments.

Techniques Used: Inhibition, Activity Assay, Cell Fractionation, Incubation, SDS Page, Autoradiography, Immunodetection, Expressing, Labeling

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Centrifugation:

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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 ( ).

Expressing:

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 * ♦
Article Snippet: .. Next, 10 μl of eluted protein from FLAG purification of yeast cells expressing either Ubp3-FLAG or Ubp3S695A -FLAG (or, as a reaction control, yeast expressing untagged Ubp3) was added along with 6 μCi of [γ-32 P]ATP (PerkinElmer Life Sciences) and incubated for 3 h at 30 °C. ..

Synthesized:

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.

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 * ♦
Article Snippet: Next, 10 μl of eluted protein from FLAG purification of yeast cells expressing either Ubp3-FLAG or Ubp3S695A -FLAG (or, as a reaction control, yeast expressing untagged Ubp3) was added along with 6 μCi of [γ-32 P]ATP (PerkinElmer Life Sciences) and incubated for 3 h at 30 °C. .. 32 P incorporation was detected by autoradiography ( ).

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.

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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 ( ).

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 * ♦
Article Snippet: .. Next, 10 μl of eluted protein from FLAG purification of yeast cells expressing either Ubp3-FLAG or Ubp3S695A -FLAG (or, as a reaction control, yeast expressing untagged Ubp3) was added along with 6 μCi of [γ-32 P]ATP (PerkinElmer Life Sciences) and incubated for 3 h at 30 °C. ..

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: 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).

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: 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.

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 * ♦
Article Snippet: .. Next, 10 μl of eluted protein from FLAG purification of yeast cells expressing either Ubp3-FLAG or Ubp3S695A -FLAG (or, as a reaction control, yeast expressing untagged Ubp3) was added along with 6 μCi of [γ-32 P]ATP (PerkinElmer Life Sciences) and incubated for 3 h at 30 °C. ..

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.

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 * ♦
Article Snippet: Paragraph title: In Vitro Kinase Assays ... Next, 10 μl of eluted protein from FLAG purification of yeast cells expressing either Ubp3-FLAG or Ubp3S695A -FLAG (or, as a reaction control, yeast expressing untagged Ubp3) was added along with 6 μCi of [γ-32 P]ATP (PerkinElmer Life Sciences) and incubated for 3 h 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 ( ).

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 * ♦
Article Snippet: Next, 10 μl of eluted protein from FLAG purification of yeast cells expressing either Ubp3-FLAG or Ubp3S695A -FLAG (or, as a reaction control, yeast expressing untagged Ubp3) was added along with 6 μCi of [γ-32 P]ATP (PerkinElmer Life Sciences) and incubated for 3 h at 30 °C. .. Samples were boiled for 5 min and resolved by SDS-PAGE and stained with Coomassie Brilliant Blue (Bio-Rad).

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  • 99
    PerkinElmer γ 32 p atp
    PXR is phosphorylated in vitro and in cells (A) His-PXR (1 or 2.5 µg) was incubated at 37°C for 30 min with Cdk2 and cyclin E along with [γ- 32 <t>P]-ATP.</t> Samples were resolved on a 4–12% gradient gel, and [γ- 32 P]-ATP incorporation was visualized using a phosphor screen (upper panel), and protein amounts in the samples were detected by SimplyBlue staining of the gel (lower panel). Histone H1 and His-tag were used as a positive and negative substrate control, respectively. The PXR band was indicated with an arrow. (B) Phosphorylation sites identified by using mass spectrometry analysis in His-PXR WT phosphorylated by Cdk2/cyclin E in vitro , and in Flag-PXR WT, Flag-PXR T133A, or Flag-PXR T135A immunoprecipitated from HEK293T cells transiently transfected with corresponding plasmid ( in vivo ). Serine or threonine residues followed by an asterisk (*) indicate phosphorylated residues; UM = unmodified peptide; M = phosphorylated peptide; nd = not detected; nt = not tested. Signal intensities are calculated from area under the curve for the detected precursor ions. (C) Anti-Flag immunoprecipitated samples prepared from HEK293T cells transiently overexpressing either Flag-PXR WT (lanes 1 2) or mutants Flag-PXR T133A (lanes 4 5) or Flag-PXR T135A (lanes 7 8) were resolved on gradient gel and stained using Sypro Ruby stain. (D) Modified peptide sequence TFDTTFS*HFK (asterisk indicating serine phosphorylation), was identified based on assignment of multiple product ions ( b and y ions) in the MS/MS scan of the precursor ion at M/z 665.78. The phosphorylation of serine 167 was confirmed based on the assignment of characteristic “ y-H 3 PO 4 ” ions and other ions (based on a mass loss of 97.9769 Da). (E) Extracted-ion chromatography (XIC) of wild type and mutant PXR sequences showing elution times and signal intensities for the non-modified peptide as well as the singly phosphorylated peptide. Panel (a) and (b) are derived from the immunoprecipitated T133A sample and show the TGAQPLGVQGLTEEQR and T*GAQPLGVQGLTEEQR, respectively. Panel (c) and (d) are derived from the immunoprecipitated T135A sample and show the AGTQPLGVQGLTEEQR and AGT*QPLGVQGLTEEQR, respectively. Panel (e) and (f) are derived from the immunoprecipitated PXR WT sample and show the TGTQPLGVQGLTEEQR and T*GTQPLGVQGLTEEQR/ TGT*QPLGVQGLTEEQR, respectively. Relative abundance (RA) of the signals of the corresponding peptides is noted for each XIC.
    γ 32 P Atp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 99/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 10 article reviews
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    γ 32 p atp - by Bioz Stars, 2020-02
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    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.
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    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