Human Phospho-BTK (Y551) Antibody

Detection of Human Phospho-BTK (Y551) by Western Blot.

Western blot shows lysates of RL human non-Hodgkin's lymphoma B cell line and Ramos human Burkitt's lymphoma cell line untreated (-) or treated (+) with 0.1 mM Pervanadate (PV) for 10 minutes and 12 µg/mL Goat Anti-Human IgM µ Chain Antigen Affinity-purified Polyclonal Antibody (Catalog # G-105-C) for 2 minutes. PVDF membrane was probed with 1 µg/mL of Mouse Anti-Human Phospho-BTK (Y551) Monoclonal Antibody (Catalog # MAB7659) followed by HRP-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # HAF018). A specific band was detected for Phospho-BTK (Y551) at approximately 75 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Detection of Human Phospho-BTK (Y551) by Simple Western^TM.

Simple Western lane view shows lysates of Ramos human Burkitt's lymphoma cell line untreated (-) or treated (+) with ultraviolet light (UV), loaded at 0.2 mg/mL. A specific band was detected for Phospho-BTK (Y551) at approximately 89 kDa (as indicated) using 20 µg/mL of Mouse Anti-Human Phospho-BTK (Y551) Monoclonal Antibody (Catalog # MAB7659). This experiment was conducted under reducing conditions and using the 12-230 kDa separation system.

Detection of Phospho-BTK (Y551) by Western Blot

Antigen-triggered phosphorylation of BTK and PLC gamma in BMMCs is independent of PI3K. (a) WT BMMCs were pre-treated for 20 min with 100 nM Wortmannin (WM) or vehicle (DMSO) and were then left untreated (con) or stimulated with the indicated concentrations of Ag (DNP-HSA [DNP]) for 1 and 5 min. Btk−/− BMMCs were pre-treated with DMSO and stimulated accordingly. Whole-cell lysates were subjected to Western Blot analysis with antibodies against P-BTK (Tyr223), P-PKB (Ser473), and p85 (loading control). (b) WT BMMCs were pre-treated for 20 min with 100 nM WM or vehicle (DMSO) and were then left untreated (con) or stimulated with Ag (20 ng/ml DNP-HSA) for the indicated times. Btk−/− BMMCs were pre-treated with DMSO and stimulated accordingly. Whole-cell lysates were subjected to Western Blot analysis with antibodies against P-BTK (Tyr551), BTK, P-PKB (Ser473), and p85 (loading control). (c) WT and Btk−/− BMMCs were pre-treated for 20 min with 100 nM WM or vehicle (DMSO) and were then left untreated (con) or stimulated with the indicated concentrations of Ag for 1 and 5 min. Whole-cell lysates were analysed by immunoblotting against P-PLC gamma1 (Tyr783), PLC gamma1, P-PKB (Ser473), and p85 (loading control). Densitometry of P-PLC gamma1 with reference to PLC gamma1 was performed using ImageJ software (NIH, USA) and normalised to WT cells pretreated with DMSO and stimulated with 1 ng/ml Ag (1 min). Comparable results were obtained in at least three experiments with different BMMC cultures. Fine vertical lines were inserted to allow for better discrimination of cells/conditions. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/30341350), licensed under a CC-BY license. Not internally tested by R&D Systems.