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Phospho btk

Manufactured by Cell Signaling Technology
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Phospho-BTK is a lab equipment product that detects and measures the phosphorylation of Bruton's Tyrosine Kinase (BTK) protein. It is used to analyze cellular signaling pathways involving BTK.

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Lab products found in correlation

Total btk, by Cell Signaling Technology (2 mentions) Total erk1 2, by Cell Signaling Technology (2 mentions) Phospho akt, by Cell Signaling Technology (2 mentions) Phospho lyn, by Cell Signaling Technology (1 mentions) Phospho gsk 3 beta, by Cell Signaling Technology (1 mentions) Phospho b raf, by Cell Signaling Technology (1 mentions) Total lck, by Cell Signaling Technology (1 mentions) Mek1 2 inhibitor u0126, by Cell Signaling Technology (1 mentions) Alamarblue cell viability reagent, by Thermo Fisher Scientific (1 mentions) Pe annexin 5 apoptosis detection kit 1, by BD (1 mentions) Vinculin clone hvin 1, by Merck Group (1 mentions) Phospho s6 ribosomal protein, by Cell Signaling Technology (1 mentions) Cleaved parp asp214 d64e10 xp, by Cell Signaling Technology (1 mentions) Ecl detection reagent, by Cytiva (1 mentions) Phospho 4ebp1, by Cell Signaling Technology (1 mentions) Cleaved caspase 3, by Cell Signaling Technology (1 mentions) Bca protein assay kit, by Thermo Fisher Scientific (1 mentions) Phospho plcγ2, by Cell Signaling Technology (1 mentions) β actin antibody, by Santa Cruz Biotechnology (1 mentions) Phospho erk1 2, by Cell Signaling Technology (1 mentions)

3 protocols using phospho btk

1

Obinutuzumab and Rituximab Signaling Analysis

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Obinutuzumab (GA101) was generously supplied by Hoffmann La Roche (Switzerland). RTX was purchased from Genentech Inc. (South San Francisco, CA, USA). Antibodies for flow cytometry, CD20-PE (Clone HI47), CD20-FITC (Clone HI47) and CD19-FITC (SJ25-C1), were obtained from Invitrogen (CA, USA). Western blot antibodies, phospho-PLC gamma2 (Tyr759, 150 KDa), total PLC gamma2 (150 KDa), phospho GSK3 Beta (Ser21/9, 46 KDa), total GSK3 Beta (46KDa), phospho BTK (Tyr223, 78KDa), total BTK (Clone D3H5, 77KDa), phospho Lyn (Tr507, 53, 56KDa) and total Lyn (C13F9,58KDa), phospho B- RAF (Ser445, 86KDa), total B- RAF (clone 55C6, 86KDa), phospho ERK1/2 (Thr202/Thyr204) (Clone D1.14.4E, 42, 44KDa), total ERK1/2 (42,44KDa), phospho LCK (Tyr505, 56KDa), total LCK (56KDa) and B-actin (clone D6A8, 45KDa) were obtained from Cell Signaling Technology (Danvers, MA, USA). CD20 (B9E9) antibody was obtained by Santa Cruz Biotechnology (Dallas, TX, USA). Annexin V: PE Apoptosis Detection Kit I was obtained from BD Biosciences (San Jose, CA, USA). Alamar blue cell viability reagent was purchased from Invitrogen (Grand Island, NY, USA). U0126 MEK1/2 inhibitor was purchased from Cell Signaling Technology (Danvers, MA, USA). PLC gamma2 and GSK3 Beta ON-TARGET plus siRNA were obtained from Dharmacon GE (Lafayette, CO, USA).
Awasthi A., Rolland D.C., Ayello J., van de Ven C., Basrur V., Conlon K., Fermin D., Barth M.J., Klein C., Elenitoba-Johnson K.S., Lim M.S, & Cairo M.S. (2017). A comparative global phosphoproteomics analysis of obinutuzumab (GA101) versus rituximab (RTX) against RTX sensitive and resistant Burkitt lymphoma (BL) demonstrates differential phosphorylation of signaling pathway proteins after treatment. Oncotarget, 8(69), 113895-113909.
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2

Western Blot Analysis of Apoptosis Markers

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Snap-frozen tissues or cell culture cells were lysed and homogenized in 1% triton lysis buffer (#9803, Cell Signaling) containing fresh protease and phosphatase inhibitors by standard procedures. Protein concentrations were quantified with the BCA Protein Assay kit (Pierce Chemical Co.), and proteins were separated in a gradient (4–15%) SDSPAGE gel, transferred to nitrocellulose membranes, and hybridized with antibodies to the indicated antigens by standard procedures. Signals were detected by chemoluminescence using ECL detection reagents (Amersham Pharmacia Biotech). Primary antibodies to the following antigens were used: cleaved PARP (Asp214/D64E10 XP, #5625, Cell Signaling), cleaved caspase-3 (Asp175, #9661, Cell Signaling), Vinculin (clone hVIN-1, V9131, Sigma), BTK (#8546, Cell Signaling), phospho-BTK (#5083, Tyr223Y223, Cell Signaling), phospho-S6 Ribosomal protein (#2211, Ser 235/236, Cell Signaling), phospho-4EBP1 (#2855, Thr37/46, Cell Signaling) and phospho-AKT (#4060, Ser473, Cell Signaling).
Grommes C., Pastore A., Palaskas N., Tang S.S., Campos C., Schartz D., Codega P., Nichol D., Clark O., Hsieh W.Y., Rohle D., Rosenblum M., Viale A., Tabar V.S., Brennan C.W., Gavrilovic I.T., Kaley T.J., Nolan C.P., Omuro A., Pentsova E., Thomas A.A., Tsyvkin E., Noy A., Palomba M.L., Hamlin P., Sauter C.S., Moskowitz C.H., Wolfe J., Dogan A., Won M., Glass J., Peak S., Lallana E.C., Hatzoglou V., Reiner A.S., Gutin P.H., Huse J.T., Panageas K.S., Graeber T.G., Schultz N., DeAngelis L.M, & Mellinghoff I.K. (2017). Ibrutinib Unmasks Critical Role of Bruton Tyrosine Kinase in Primary CNS Lymphoma. Cancer discovery, 7(9), 1018-1029.
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3

Ibrutinib Inhibits BCR Signaling

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Jeko-1 and MCIR1 cells were incubated with vehicle or 1.0 μM ibrutinib (Chemitek) for 30 minutes, and subsequently stimulated for 30 minutes with 5ug/mL anti-IgA, anti-IgG and anti-IgM cocktail whilst still in the presence of ibrutinib. Cells were lysed in RIPA buffer, the lysates were resolved by SDS-PAGE, transferred to PVDF membranes, and probed with the following antibodies: phospho-BTK, total BTK, phospho-PLCγ2, total PLCγ2, phospho-AKT, total AKT, phospho-ERK1/2, and total ERK1/2 from Cell Signaling (Danvers, MA); and β-actin antibodies from Santa Cruz Biotechnology (Santa Cruz, CA). Images were developed either on a Li-COR imager or on X-ray films using SuperSignal kit.
Wu X., Nowakowski K.E., Abeykoon J.P., Manske M., Stenson M.J., Timm M.M., Hanson C.A., Dyke D.L., Dasari S, & Witzig T.E. (2021). MCIR1: A Patient-Derived Mantle Cell Lymphoma Line for Discovering New Treatments for Ibrutinib Resistance. European journal of haematology, 107(4), 458-465.
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