Ba/F3 cells expressing Bcr-AblWT and Bcr-AblT315I were obtained from Dr. Shah’s laboratory (UCSF, San Francisco, CA, USA). The cells were maintained in RPMI 1640 (Corning) supplemented with 10% fetal bovine serum (v/v, HyClone, ThermoScientific) and 1× penicillin, strepatmycin, and glutamine (GE Healthcare) at 37 °C and 5.0% CO2. Imatinib and ponatinib were purchased from Selleckchem. Dimethyl sulfoxide (DMSO) was purchased from Sigma-Aldrich (St. Louis, MO, USA). The following antibodies were purchased from Cell Signaling (Boston, MA, USA): anti-Abl, anti-phospho-Abl (Y412), anti-phospho-STAT5 (Tyr694), and anti-STAT5. Anti-actin antibody was purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-mouse secondary antibody was purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA), whereas anti-rabbit secondary antibody was obtained from Abcam (Cambridge).
Anti phospho stat5 tyr694
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-phospho-STAT5 (Tyr694) is a monoclonal antibody that specifically recognizes the tyrosine 694-phosphorylated form of the Signal Transducer and Activator of Transcription 5 (STAT5) protein. STAT5 is a transcription factor that plays a crucial role in cell signaling pathways.
Automatically generated - may contain errors
Lab products found in correlation
Anti stat5, by Cell Signaling Technology (4 mentions)
Anti jak2, by Cell Signaling Technology (2 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Imatinib, by Selleck Chemicals (1 mentions)
Penicillin, by GE Healthcare (1 mentions)
Glutamine, by GE Healthcare (1 mentions)
Ponatinib, by Selleck Chemicals (1 mentions)
Anti rabbit secondary antibody, by Abcam (1 mentions)
Hyclone, by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Anti actin antibody, by Merck Group (1 mentions)
Rpmi 1640, by Corning (1 mentions)
Anti stat1, by Cell Signaling Technology (1 mentions)
Anti phospho stat1 tyr701, by Cell Signaling Technology (1 mentions)
Anti phospho mek1 2 ser217 221, by Cell Signaling Technology (1 mentions)
Anti β actin ab6276, by Abcam (1 mentions)
Anti stat5 sc 835, by Santa Cruz Biotechnology (1 mentions)
Newblot nitrocellulose stripping buffer, by LI COR (1 mentions)
Anti α tubulin, by Cell Signaling Technology (1 mentions)
Mini protean precast gel, by Bio-Rad (1 mentions)
6 protocols using anti phospho stat5 tyr694
1
Bcr-Abl Inhibitors in Leukemia
2
Western Blot Analysis of JAK2 Signaling
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Leukemic cells were lysed in lysis buffer supplemented with freshly added protease and phosphatase inhibitors. 25μg (BCA method; Thermo Scientific) lysate was loaded on 10% mini protean precast gels (BioRad, Veenendaal, Netherlands), and transferred to a nitrocellulose membrane (Biorad). Primary antibody incubation was performed according to manufacturer's protocol. Anti-JAK2 (#3230), anti-phospho-JAK2-Tyr1007 (#4406), anti-phospho-STAT5-Tyr694 (#9351), anti-phospho-STAT1-Tyr701 (#9167), anti-Stat1 (#9175), anti-phospho-MEK1/2-Ser217/221 (#9154), anti-MEK1/2 (#4694), anti-phospho-Erk1/2-T202/204 (#4370), anti-Erk1/2 (#91078), and anti-αTubulin (#2144) were obtained from Cell Signaling Technology (Danvers, Massachusetts, USA). Anti-β-actin (ab6276) was obtained from Abcam (Cambridge, UK), and anti-STAT5 (sc-835) from Santa Cruz (Heidelberg, Germany). Blots were stained with secondary antibodies (IRDye 680RD- or 800CW-labelled anti-rabbit and IRDye 680RD- or 800CW-labelled anti-mouse; Li-Cor Biosciences, Leusden, Netherlands) and scanned using the Odyssey infrared imaging system (Li-Cor Biosciences). To reprobe membranes, they were stripped in NewBlot Nitrocellulose stripping buffer (Li-Cor Biosciences) according to manufacturer's protocol. BCR-JAK2, PAX5-JAK2 and TERF2-JAK2 proteins were separated from wildtype JAK2 based on size (~94, 57, 95 and 125 kDa, respectively).
3
Profiling Kinase Signaling in K562 Cells
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Proteins were extracted from K562 cells cultured with DMSO, 1.0 μM daphnoretin, 0.5 μM midostaurin (a PKC inhibitor), or TPA at 0.1 μM for 24, 48, and 72 h using radioimmunoprecipitation assay (RIPA) lysis buffer (Sigma Aldrich, St. Louis, MI, USA) with protease and phosphatase inhibitors (Roche, Indianapolis, IN, USA). Western blotting was performed according to a previous study [32 (link)]. Membranes were incubated overnight at 4 °C with the following primary antibodies at a 1:1000 dilution: anti-JAK2, anti-phospho-JAK2 (Tyr1007/1008), anti-STAT5, anti-phospho-STAT5 (Tyr694), anti-phospho-STAT3 (Tyr705) antibody (all from Cell Signaling Technology, MA, USA), and anti-STAT3 antibody (BD Biosciences, San Jose, CA, USA). Protein expression was detected using the ChemiGenius Bio Imaging System (Syngene, Frederick, MD, USA). The intensity of the bands was quantified using ImageJ software and levels were normalized to those of the internal control, β-actin.
4
STAT5 Phosphorylation Analysis Protocol
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To analyze STAT5 phosphorylation, cells were lysed with NETN extract buffer containing 100 mM NaCl, 20 mM Tris-Cl (pH 8), 0.5 mM EDTA, 0.5% Nonidet P-40, protease inhibitor cocktail (cOmplete; Roche), and phosphatase inhibitor cocktail (phosSTOP; Roche) for 30 min on ice. Lysates were cleared by centrifugation at 12,000 rpm for 10 min at 4°C. Proteins were visualized by SDS-PAGE. Western blotting was performed according to standard protocols. The following antibodies were used at the following concentrations: anti-STAT5 at a 1:1,000 dilution (clone D2O6Y; Cell Signaling Technology, Danvers, MA), anti-phospho STAT5 (Tyr694) at a 1:1,000 dilution (clone C11C5; Cell Signaling Technology), and anti-β-actin antibody at a 1:10,000 dilution (clone AC-15; Sigma-Aldrich, St. Louis, MO). Secondary anti-rabbit and secondary anti-mouse antibodies (Jackson ImmunoResearch, catalog no. 111-035-046 and 115-035-146, respectively) were used at a 1:10,000 dilution.
5
Signaling Pathway Analysis via Western Blot
6
Western Blot Analysis of Protein Expression
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Cells were lysed using RIPA buffer (Sigma-Aldrich) supplemented with 1 × Halt Protease and Phosphatase Inhibitor cocktail (Thermo Fisher Scientific). Lysates were subjected to 10% SDS-PAGE followed by transfer to nitrocellulose membrane (Bio-Rad). Membranes were blocked with 5% nonfat dry milk and incubated with primary antibodies overnight at +4°C followed by incubation with secondary horseradish peroxidase–conjugated antibodies for 2 hours at room temperature. The following primary antibodies were used: anti-His-tag (RRID: AB_2744546), anti-S100A9 (RRID: AB_2734726), anti-phospho-STAT5 (Tyr694) (RRID: AB_10544692), anti-STAT5 (RRID: AB_2737403; Cell Signaling Technology), anti-β-actin (Santa Cruz Biotechnology, RRID: AB_626632).
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