Anti c cbl antibody

Cell lysates were prepared in lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% (v/v) Nonidet P-40, 0.5 mM EDTA) supplemented with complete protease inhibitor cocktail (Roche,04693132001). Soluble protein was subjected to immunoprecipitation with anti-Flag antibody (agarose-conjugated, M-2, Sigma-Aldrich, A2220), anti-c-Abl antibody (K-12, Santa Cruz Biotechology, Cat# SC-131), or anti-rabbit IgG antibody (agarose-conjugated, Sigma-Aldrich). An aliquot of the total lysate (5%, v/v) was included as a control. Immunoblot analysis was performed with anti-HAX-1 antibody (FL-279, Santa Cruz Biotechology, sc-28268), anti-c-Abl antibody (K12 rabbit polyclonal antibody, Santa Cruz), anti-c-Cbl antibody (Santa Cruz, sc-1651), anti-tubulin antibody (Sigma-Aldrich, Cat# T9822), anti-β-actin(Santa Cruz,Cat# SC-1616), or HRP conjugated anti-Flag antibody (Sigma-Aldrich, Cat# A8592), anti-Myc antibody (Santa Cruz, Cat# SC-40), anti-Ub antibody (Biomol, NSJ-R30490) and anti-phosphotyrosine antibody (4G10, Millipore, Cat# 16-105). The antigen-antibody complexes were visualized by chemiluminescence (ECL, Millipore, WBKLS0500). Data shown are representative of three independent experiments. The relative intensity of WB bands was quantified by gray scanning and represented as mean±S.D. of three independent analysis. n.s., not significant; *p < 0.05, **p < 0.01, ***p < 0.001, Student’s t test.

Anti-Src antibody was obtained from Merck Millipore. Anti-phosphotyrosine (pTyr-100) antibody, anti-pY731 c-Cbl antibody, and anti-phospho-cortactin antibody (pY421) were obtained from Cell Signaling Technology. Anti-c-Cbl antibody was obtained from Santa Cruz Biotechnology.