Recombinant EGFR (#3641, Sigma-Aldrich, MO), purified recombinant GST-MOB1 protein, 200 μM ATP (#9804, Cell Signaling Technology, MA), 1× kinase buffer (#9802, Cell Signaling Technology, MA) were mixed and incubated at 30 °C for 30 min. The reaction was stopped by adding sample buffer, and incubated at 95 °C for 5 min. For LATS1 kinase activity, HEK293 cells were harvested and used for immunoprecipitation as described above. Primary antibody against IgG or LATS1 were used. After washing the beads, the beads were incubated with recombinant YAP (ab132459, Abcam, CA), 200 μM ATP (#9804, Cell Signaling Technology, MA), 1× kinase buffer (#9802, Cell Signaling Technology, MA) at 30 °C for 30 min. The reaction was stopped by adding sample buffer, then incubated at 95 °C for 5 min. The beads were centrifuged and removed, and supernatant was used for western blot.
Adenosine triphosphate (atp)
Manufactured by Cell Signaling Technology
Sourced in United States, Morocco
ATP is a nucleotide that serves as the primary energy currency in living cells. It is involved in the transfer of energy within cells, playing a crucial role in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Kinase buffer, by Cell Signaling Technology (19 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (4 mentions)
Gsk 3 fusion protein peptide, by Cell Signaling Technology (2 mentions)
Lysis buffer, by Cell Signaling Technology (2 mentions)
Phospho tracer elisa kits, by Abcam (2 mentions)
Kinase assay buffer, by Cell Signaling Technology (2 mentions)
Kinase reaction buffer, by Cell Signaling Technology (2 mentions)
Phosphotracer elisa kit, by Abcam (2 mentions)
Recombinant active cdk5 p25 and cdk5 p35 proteins, by Merck Group (2 mentions)
Active tau protein, by AnaSpec (2 mentions)
Active creb1, by Thermo Fisher Scientific (2 mentions)
γ 32p atp, by PerkinElmer (2 mentions)
Sc 108080, by Santa Cruz Biotechnology (1 mentions)
Claudin 5, by Cell Signaling Technology (1 mentions)
U73122, by Bio-Techne (1 mentions)
Pfgfr2, by Cell Signaling Technology (1 mentions)
Neb 5 alpha competent e coli high efficiency, by New England Biolabs (1 mentions)
Cd 63 antibody, by GeneTex (1 mentions)
Total exosome rna and protein isolation kit, by Thermo Fisher Scientific (1 mentions)
Total erk, by Cell Signaling Technology (1 mentions)
42 protocols using adenosine triphosphate (atp)
1
Kinase Assay for EGFR and LATS1 Activity
2
Detailed Molecular Signaling Pathway Protocol
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FGF9 (catalogue # 273-F9-025) was purchased from R&D Systems. METAFECTENE® PRO (#T040) was purchased from Biontex. TRPA1 antibodies were purchased from Alomone (#ACC-037) and Santa-Cruz (#sc-166469). Total FGFR2 antibody (#sc-122) was purchased from Santa-Cruz. pFGFR2 (#3471), pERK (#4370), Total ERK (#9102) β-actin (#4970), Flag (#8146), Ki-67 (#9449), β-tubulin (#2146), Zona Occludin-1 (#5406), Claudin-5 (#4933), IgG antibody (#2729), anti-GFP (#2956) antibodies along with U0126 (#9903) were purchased from Cell Signalling. U73122 (#1268) was purchased from TOCRIS. NEB 5-alpha competent E.Coli (high efficiency) (#C2987H) cells purchased from New England Biolabs, were used for transformation. TRPA1 inhibitor HC030031 (#2896) was purchased from Tocris bioscience. ATP (#9804) and kinase buffer (#9802) were purchased from Cell Signalling. Occludin (H-279) (sc-5562) antibody, validated shRNAs18 (link), 55 (link) (sc-29218-V, sc-108080, and sc-29452-V), and DMA (sc-202459) were purchased from Santa Cruz Biotechnology, Inc. Strep antibody (# ab76949) was bought from abcam. CD-63 antibody (#GTX41877) from GeneTex was utilized as an exosomal marker (exosomal proteins were extracted using the Total Exosome RNA and Protein Isolation kit (#4478545, Invitrogen). All antibodies were used in 1:1000 dilutions for western blots.
3
In Vitro Phosphorylation of ATG4B by MST4
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The pcDNA3.1 expression vectors of myc-MST4-WT and –K53E were transfected into HEK293T cells by using lipofectamine 2000 (Thermo Fisher Scientific). After 48 hr, cells were lysed in lysis buffer containing 20 mM Tris-HCl (pH 8.0), 0.5% Nonidet P-40, 250 mM NaCl, 3 mM EDTA, 3 mM EGTA, 2 mM DTT, 0.5 mM phenylmethylsulfonyfluoride (PMSF), 20 mM beta-glycerolphosphate, 1 mM sodium orthovanadate and 1 μg/ml leupeptin. Myc-MST4 protein in cell lysates was immunoprecipitated by using a mouse anti-Myc-Tag antibody (9B11) conjugated with Sepharose beads (Cell Signaling, # 3400) by rotation at 4°C overnight. The precipitated MST4 proteins were re-suspended in 40 μl of 1 X kinase buffer (Cell Signaling, #9802) supplemented with 200 μM ATP (Cell Signaling, #9804) and purified His-ATG4B-WT or –S383A protein, which was expressed in pET-28a vector in E. coli. The reaction was carried out for 30 min at 30°C, and was terminated with 20 μl 3X SDS sample buffer. Each sample was then boiled for 10 min at 100°C, followed by SDS-PAGE and IB analysis using indicated antibodies.
4
Kinase Activity Assay for Ezrin and Caveolin-1
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In vitro kinase assay was performed as described previously [25 (link)]. In brief, Escherichia coli expressed GST fusion proteins (GST-ezrin, GST-caveolin-1) were purified by GSTrap FF Columns (GE Healthcare, IL, USA), cleaved for GST portion by thrombin (Sigma Aldrich), and further purified by HiTrap Benzamiidine FF Columns (GE Healthcare) according to the manufacturer’s instructions. 2μg of these polypeptides were incubated with commercial Src (active, 0.5 μg, Abcam) and ATP (20 μM, Cell Signaling Technology) in kinase buffer (Cell Signaling Technology), with or without PP2 (1 μM), for 30 min at 37°C. The reaction was stopped by adding SDS-PAGE loading buffer and boiling. Phosphorylated ezrin or caveolin-1 was detected by western blotting.
5
Evaluating mTOR Kinase Inhibition
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The kinase assay was performed according to the manufacturer’s instructions (Promega, Madison, WI, USA). Briefly, the active recombinant mTOR (50 ng) protein was mixed with different concentrations of ethyl ferulate in reaction buffer and incubated at room temperature for 15 min. Next, the inactive p70S6K recombinant protein (100 ng) and ATP (Cell Signaling Technology) were added and incubated for 30 min at 30°C. The reaction was stopped by adding 10 μl protein loading buffer. The protein band was detected by Western blot analysis. mTOR activity was assessed using a p70S6K phosphorylation antibody.
6
Kinase Activity Assay for CK2α
7
Kinase Assay for AKT1/2 Variants
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MCF10A cells expressing empty vector, HA-AKT1/2, or HA-AKT1/2(E17K) were serum starved for 16 hours and lysed as described above in EBC buffer (0.5% NP-40, 120 mmol/L NaCl, 2 mmol/L EDTA, 2 mmol/L EGTA, 50 mmol/L Tris---HCl (pH 7.4), proteinase inhibitor cocktail, 50 nmol/L calyculin, 1 mmol/L sodium pyrophosphate, and 20 mmol/L sodium fluoride). HA-AKT1/2 or HA-AKT1/2(E17K) were immunoprecipitated from cell extracts with an anti-HA antibody and incubated with 300 ng GSK-3 fusion protein peptide (Cell Signaling Technology) in the presence of 150 mmol/L cold ATP in a kinase buffer (Cell Signaling Technology) for 40 min at 30°C. The kinase reaction was terminated by the addition of SDS-PAGE sample buffer.
8
TAK1 Kinase Assay with EZH2
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TAK1 in vitro kinase assays were carried out as previously described (Wan et al, 2018 (link)). In brief, recombinant human EZH2 protein (Novus Biologicals, H00002146‐P01) was incubated with recombinant TAK1 protein (Novus Biologicals, H00006885‐P01) in kinase buffer (Cell Signaling, 9802S), supplemented with 300 µM ATP (Cell Signaling, 9804) for 60 min at 30°C. The reaction was stopped by adding 5× fluorescent loading buffer, followed by WB analysis.
9
In Vitro Kinase Assay Protocol
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For in vitro kinase assays, 1–2 μg substrate were incubated with 0.25–0.5 μg activated kinase in kinase buffer (50 mM Tris-Cl, pH 7.5, 0.1 mM EGTA, 1 mM DTT, 7.5 mM Mg[CH3COO]2, 2 µM ATP [non-radioactive; Cell Signaling Technology, 9804] with or without 10 μCi [γ-32P]-ATP [Hartmann Analytics, SRP 301]) for 45 min at 30°C. The kinase reaction was stopped by adding loading buffer and boiling for 5 min at 95°C. After Coomassie Brilliant Blue staining or immunoblotting, autoradiography was performed or phosphorylation was detected using anti-PRKAA1/2 phospho-T183/T172 antibody.
10
GSK3β Kinase Activity Assay
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For in vitro kinase assay, 1 µg recombinant GSK3β was incubated with 200 ng active eEF2K in 1× kinase buffer (Cell Signaling Technology, 9802) supplemented with ATP (Cell Signaling Technology, 9804) at a final concentration of 200 μM. After incubation for 1 hour at 30°C, the reaction was terminated by the addition of SDS-PAGE sample loading buffer and followed by western blot analysis.
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