Anti pstat5 tyr694

Manufactured by Cell Signaling Technology

Sourced in United States

Anti-pSTAT5 (Tyr694) is a research-use-only primary antibody that specifically recognizes the phosphorylated form of STAT5 protein at tyrosine 694. It can be used to detect the activation of STAT5 signaling pathways in biological samples.

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5 protocols using anti pstat5 tyr694

Immunofluorescence analysis of mammary gland

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Dissected mammary fat pads were fixed in MethaCarn and embedded in paraffin. Seven μm-thick sections were deparaffinized before staining with primary antibodies (overnight, 4°C), and secondary antibodies (1 h, room temperature). Nuclei were counterstained with DAPI. Primary antibodies used were: rabbit polyclonal anti-PAR3 (1:200; Chemicon), anti-aPKC (1:200; clone C-20, Santa Cruz Biotechnology), anti-RAB11A (1:200; Abcam), anti-pSTAT5 (Tyr694, 1:100; Cell Signalling), anti-cleaved caspase 3 (1:100; Cell Signalling), anti-WAP (1:300; clone R-131, Santa Cruz Biotechnology) and anti-keratin 5 (K5) (1:2,000; Covance); rabbit monoclonal anti-KI67 (1:100; clone SP6, Neo Markers); and mouse monoclonal anti-HTT (1:300; 4C8), anti-E-cadherin (1:200; BD Bioscience) and anti-GM130 (1:100; BD Bioscience). Antigen retrieval was performed by boiling the slides for 10 min in a microwave in 10 mM citrate buffer (pH 6) for cleaved caspase 3, Ki67, WAP, and p-STAT5A, or in EDTA buffer (pH 8.8) for 10 min for PAR3, aPKC, RAB11A, HTT, GM130, K5, and E-cadherin antibodies. Secondary antibodies used were goat anti-mouse and anti-rabbit conjugated to AlexaFluor-488 or AlexaFluor-555 or Biotin (Vector Laboratories).

Elias S., McGuire J.R., Yu H, & Humbert S. (2015). Huntingtin Is Required for Epithelial Polarity through RAB11A-Mediated Apical Trafficking of PAR3-aPKC. PLoS Biology, 13(5), e1002142.

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Immunoblotting and Co-Immunoprecipitation Protocol

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Immunoblotting and co-IP were performed as described previously (3 (link)). Briefly, for reducing conditions, 8% β-mercaptoethanol was added into 3× SDS sample buffer [75 mmol/L Tris (pH 6.8), 3% SDS, 15% glycerol, and 0.1% bromophenol blue]. The mixed samples were boiled for 5 minutes in a 95°C heat block before loading on a 4% to 15% Tris-HCl gradient gel (Bio-Rad). For nonreducing conditions, no β-mercaptoethanol was added, and the nonboiled samples were loaded on a 10% gel. The following primary antibodies and reagents were used: anti-GCSF receptor antibody (Abcam, ab126167), anti-GFP antibody (Santa Cruz Biotechnology, sc-9996), anti-pSTAT3 Tyr705 (Cell Signaling Technology, 9131), anti-STAT3 (Cell Signaling Technology, 9132), anti-pSTAT5 Tyr694 (Cell Signaling Technology, 9151), anti-STAT5 (BD Transduction Laboratories, 610192), anti-pERK1/2 Thr202/Tyr204 (Cell Signaling Technology, 4370), anti-ERK1/2 (Cell Signaling Technology, 9102), anti-pJAK2 Tyr1007/1008 (Cell Signaling Technology, 3776), anti-JAK2 (Cell Signaling Technology, 3230), and HRP conjugate secondary antibodies against mouse IgG and rabbit IgG (Promega).

Zhang H., Means S., Schultz A.R., Watanabe-Smith K., Medeiros B.C., Bottomly D., Wilmot B., McWeeney S.K., Kükenshöner T., Hantschel O, & Tyner J.W. (2017). Unpaired Extracellular Cysteine Mutations of CSF3R Mediate Gain or Loss of Function. Cancer research, 77(16), 4258-4267.

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Western Blot Analysis of Signaling Proteins

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Western blot analysis was performed as described previously.^{22 (link)} The following antibodies were used: anti-p-ERK (T202/Y204) (Cell Signaling Technology, Danvers, MA, USA), anti-ERK (Cell Signaling Technology; 9102), anti-p-AKT (Ser473) (Cell Signaling Technology; 9271), anti-AKT (Cell Signaling Technology; 9272), anti-p-Stat5 (Tyr694) (Cell Signaling Technology; 9351), anti-Stat5 (Cell Signaling Technology; 9363), anti-p38 (Cell Signaling Technology; 9212), anti-p-p38 (Tyr180/182) (Cell Signaling Technology; 9216), anti-Mcl-1 (Cell Signaling Technology; 4572), and anti-GAPDH (Cell Signaling Technology; 5174).

Wang X., Pan B., Honda G., Wang X., Hashimoto Y., Ohkawara H., Xu K., Zeng L, & Ikezoe T. (2018). Cytoprotective and pro-angiogenic functions of thrombomodulin are preserved in the C loop of the fifth epidermal growth factor-like domain. Haematologica, 103(10), 1730-1740.

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Western Blot and Immunostaining Reagents

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Primary antibodies used in western blot studies included anti-MYC (Y69, #ab32072), anti-Sox2 (EPR3131; #ab92494) and anti-Oct4 (#ab19857), which were purchased from Abcam (Cambridge, MA, USA), anti-β-actin (#sc-47778) from Santa Cruz Biotechnology (Santa Cruz, CA, USA), anti-FLT3 (8F2, #3462) from Cell Signaling (Danvers, MA, USA), and anti-p-STAT5 (Tyr694, #9359) from Cell Signaling. Additionally, anti-myeloperoxidase (A1F4; #MA5-42397) from Invitrogen (Waltham, MA, USA) and the anti-MYC mentioned above were used in immunostaining experiments. Gilteritinib (ASP2215, #S7754) was purchased from Selleckchem (Houston, TX, USA), and Ara-C (PHR1787) was purchased from Sigma-Aldrich (St Louis, MO, USA). Venetoclax was gifted by Abbvie. Short hairpin RNA (shRNA) plasmid for Myc was purchased from MilliporeSigma (Burlington, MA, USA). Myc overexpression vector was purchased from Addgene (#190618) (Watertown, MA, USA).

Lai J., Shang C., Chen W., Izevbaye I., Chu M.P., Sandhu I., Brandwein J., Lai R, & Wang P. (2023). An In Vitro Model for Acute Myeloid Leukemia Relapse Using the SORE6 Reporter. International Journal of Molecular Sciences, 25(1), 496.

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FLT3 Inhibitor Characterization and Kinase Assay

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The FLT3 inhibitors, BPR1J-340 and AC220, were synthesized by our laboratory. The histone deacetylase inhibitor vorinostat (SAHA) was purchased from SelleckBio (Houston, TX, USA). All inhibitors were dissolved in dimethylsulfoxide (DMSO) at a stock concentration of 10 mM. The anti-FLT3 (sc-480, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-pFLT3-Tyr591 (#3461, Cell Signaling Technology, Beverly, MA, USA) anti-STAT5 (#9363, Cell Signaling Technology), anti-pSTAT5–Tyr694 (#9351, Cell Signaling Technology), anti-cleaved poly ADP-ribose polymerase (PARP) (#9542, Cell Signaling Technology), anti-Mcl-1 (#4572, Cell Signaling Technology), anti-caspase 3 (#9662, Cell Signaling Technology) and anti-β-actin (Gtx110546, GeneTex, Irvine, CA, USA) antibodies were purchased for Western blotting analysis. The preparation of recombinant proteins, FLT3 (residues Y567-S993), VEGFR1 (residues R781-I1338) and VEGFR2 (residues V789-V1356), for biochemical kinase assay was described previously [29] (link). The VEGFR3 (residues M800-Y1363) proteins were purchased from Upstate (Billerica, MA, USA).

Lin W.H., Yeh T.K., Jiaang W.T., Yen K.J., Chen C.H., Huang C.T., Yen S.C., Hsieh S.Y., Chou L.H., Chen C.P., Chiu C.H., Kao L.C., Chao Y.S., Chen C.T, & Hsu J.T. (2014). Evaluation of the Antitumor Effects of BPR1J-340, a Potent and Selective FLT3 Inhibitor, Alone or in Combination with an HDAC Inhibitor, Vorinostat, in AML Cancer. PLoS ONE, 9(1), e83160.

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