Wild-type Flag-PES1 and mutant Flag-PES1 (S424A) proteins were translated in vitro following the manufacture protocol of TNT® Quick coupled Translation System Technical (Promega, Cat No. TM045) as described previously [22 (link)]. These proteins were purified with Pierce Protein G Agarose and primary antibody (Flag-tag antibody, Cat No. A5712, Bimake) in the cold room overnight. Then, the purified proteins were added into kinase assay buffer (Cat No.ab189135, Abcam), and incubated with activated CDK5/p25 (Cat No. ab60761, Abcam) and 50 μM ATP-γ-S (Cat No. ab138911, Abcam) at 30 °C for 45 min. 2.5 mM PNBM/5% DMSO were added to the sample at the room temperature for 1 h. The phosphorylated protein was detected by an anti-thiophosphate ester antibody (Cat No. ab92570, Abcam) [23 (link)].
Anti thiophosphate ester antibody
Manufactured by Abcam
Anti-thiophosphate ester antibody is a laboratory product designed to detect the presence of thiophosphate esters in various samples. It serves as a tool for researchers to identify and quantify these specific chemical modifications.
Automatically generated - may contain errors
Lab products found in correlation
Atp γ s, by Abcam (2 mentions)
Atpγs, by Biolog (2 mentions)
Recombinant pkcλ ι, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Dulbecco s modified eagle medium, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Anti flag affinity gel, by Merck Group (1 mentions)
Myc trap agarose, by Proteintech (1 mentions)
Polyethyleneimine pei, by Polysciences (1 mentions)
Freestyle 293 expression medium, by Thermo Fisher Scientific (1 mentions)
In fusion, by Takara Bio (1 mentions)
Anti flag m2 antibody, by Merck Group (1 mentions)
Anti flag beads, by Merck Group (1 mentions)
Anti ha beads, by Merck Group (1 mentions)
Anti phosphorylated tbk1, by Cell Signaling Technology (1 mentions)
N6 furfuryl atpγs, by Biolog (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Abcam (1 mentions)
Atpγs, by Merck Group (1 mentions)
9 protocols using anti thiophosphate ester antibody
1
PES1 Phosphorylation by CDK5/p25
2
Analog-sensitive Cdk2 kinase assay
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To generate an analog-sensitive (AS) version of Cdk2, we introduced a mutation in mouse Cdk2 cDNA that changes phenylalanine 80 to a glycine as described previously [47 (link)]. Human embryonic kidney 293T cells in 6-well plates were co-transfected using lipofectamine 2000 (Invitrogen) with plasmids encoding Flag-tagged Lin37, Mybl1 or Dmrtc2 and AS Cdk2 or wild-type Cdk2 with cyclin E1. After two days, cells were washed with PBS and incubated in the wells for 20 min at room temperature with 200 μl of a kinase reaction buffer [20 mM HEPES pH 7.5, 100 mM KOAc, 5 mM NaOAc, 2 mM MgOAc2, 1 mM EGTA, 10 mM MgCl2, 0.5 mM DTT, 30 μg/ml digitonin, 5 mM GTP, 0.1 mM ATP, 0.1 mM N6-(phenethyl) ATPγS (Biolog), 1X phosphatase inhibitor cocktail I and II (Sigma), and 1X complete protease inhibitors, EDTA-Free (Roche)], as described previously [26 (link)]. After the labeling step, 200 μl of 2x RIPA buffer (100 mM Tris pH 8.0, 300 mM NaCl, 2% NP-40, 0.2% SDS, 20 mM EDTA) with 2.5 mM p-nitrobenzyl mesylate (PNBM; Abcam) was added, and samples were incubated for 30 min at room temperature (RT). Mybl1 and Dmrtc2 were immunoprecipitated using anti-Flag antibody-coupled resin (Sigma) and the phosphorylation was detected by western blotting with an anti-thiophosphate ester antibody (Abcam).
3
In Vitro Kinase Assay for Cyclin D3
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Nonradioactive assays were performed as described previously (Allen et al., 2007 (link)). In brief, 293T cells were transfected with p3xFLAG-CMV7.1-Cyclin D3 WT or T283A and lysed 2 d later for immunoprecipitation with anti-FLAG affinity gel (Sigma-Aldrich). Bead-bound Cyclin D3 immunoprecipitates were washed extensively, and then added to kinase reactions containing a recombinant active fragment of hDYRK1A (Millipore), ATP-γS (Abcam), and kinase assay buffer (40 mM Tris, pH 7.5, 10 mM MgCl2, and 50 mM NaCl). Reactions were incubated on a rotator at room temperature for 45 min, before adding 25 mM p-nitrobenzyl mesylate (PNBM; Abcam) for 2 h to alkylate thiophosphates. Reaction products were subjected to Western blot and probed with an anti-thiophosphate ester antibody (51–8; Abcam) to visualize phosphorylated proteins.
4
HEK293 Cell Culture Protocol
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HEK293T cells were grown in Dulbecco’s modified eagle medium supplemented with 10% (v/v) fetal bovine serum (ThermoFisher Scientific), 100 U/ml penicillin, and 100 μg/ml streptomycin (ThermoFisher Scientific) at 37 °C in 5% CO2. HEK293FS cells were grown in Freestyle 293 Expression Medium (ThermoFisher Scientific) in a shaking incubator at 37 °C in 8% CO2. Cells were obtained from the institute’s central catalog, where they are routinely checked for mycoplasma. Anti-thiophosphate ester antibody was from Abcam. Anti-FLAG M2 antibody and FLAG-agarose resins were purchased from Sigma. Strep-Tactin II agarose was from GE HealthCare. Myc-trap agarose was from Chromotek. Anti-Myc, secondary HRP-linked goat anti-Rabbit, and Horse anti-Mouse antibodies were from Cell Signaling Technologies. GST-tagged Src was from Carna Biosciences. Polyethyleneimine (PEI) was from Polysciences Inc. Mutagenesis and cloning were done using In-Fusion (Takara). Primers and plasmids used are listed in Table S4 . Peptides used were made in-house.
5
Kinase Substrate Phosphorylation Assay
6
Analog-sensitive CDK Interactome Profiling
7
Kinase Assay for TBK1 and IRF3
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293T cells were transfected with the Myc-TBK1, Flag-eGFP-IRF3, HA-SAV1, wild-type, or kinase-dead HA-Mst1 expression plasmids. Thirty-six hours after transfection, cells were lysed in MLB buffer, and immunoprecipitation was performed with anti-Myc, anti-Flag, or anti-HA antibody. Beads were washed three times with MLB buffer and once with kinase assay buffer (20 μM ATP or ATPγS, 20 mM Tris-HCl, 1 mM EGTA, 5 mM MgCl2, 0.02% 2-mercapto-ethanol, 0.03% Brij-35, 0.2 mg/mL BSA). Immunoprecipitated Myc-TBK1 or Flag-eGFP-IRF3 with or without wild-type or kinase-dead HA-Mst1 (with HA-SAV1) was incubated in kinase assay buffer for 60 min at 30°C on a Thermo-Shaker. EDTA and PNBM (Abcam) were then added, and the reaction was incubated for 30 min at 25°C with mild shaking. The reaction was stopped by addition of 2× SDS loading buffer and subjected to SDS-PAGE and immunoblotting with the indicated antibodies. The integration of γ-S was detected by immunoblotting with anti-thiophosphate ester antibody (Abcam).
8
In Vitro Kinase Assay for PKC Substrate Phosphorylation
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This method has been adapted from [44 (link)]. For each reaction, 150 ng of recombinant substrate (GST-fusion proteins) was incubated with 140 ng of human recombinant PKCiota or PKCz produced in Sf9 cells (Moscat laboratory) in phosphorylation buffer {(35 mM Tris HCl pH 7.5, 10 mM MgCl2, 0.1 mM CaCl2, 0.5 mM EGTA, 1 mM DTT and 0.1 mM ATPγS (Biolog)}) for 1 hour at 30 °C. The reaction was stopped by adding 20 mM EDTA and samples were incubated with 2.5 mM PNMB (Abcam) for 1 hour at room temperature. The samples were analysed by SDS-PAGE and western blotting using anti-thiophosphate ester antibody (Abcam, 1:5000). For MS/MS detection of Nuf phosphopeptides, an in-vitro kinase assay was performed using 10 μg of GST-Nuf FL and 10 μg of PKCiota in 400uM ATP and 1x phosphorylation buffer. Phosphorylation products were digested and processed using LS-MS/MS analysis by the SBMRI Proteomic Service.
9
In Vitro Phosphorylation of SOX2
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