Horseradish peroxidase anti mouse

The western blots were performed as previously described [35 (link)]. The antibodies used for western blots: anti- CD38 was from BD Biosciences (San Jose, CA), GAPDH, c-Raf, Phospho-c-Raf (S259), Mek, p-Mek, Erk, p-Erk, FAK, Lyn, Fgr, p47phox, Slp-76, Vav1, pY416-c-Src, horseradish peroxidase anti-mouse, and anti-rabbit antibodies, were from Cell Signaling (Danvers, MA), p-Tyr and c-Cbl antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA), Phospho-c-Raf (S621) was from Thermo Fisher Scientific (Waltham, MA).

2×10⁷ cells were lysed using 350–400 µL lysis buffer (Pierce, Rockford, IL) supplemented with protease and phosphatase inhibitors (Sigma, St. Louis, MO), and lysates were cleared by centrifugation at 13,000 rpm for 30 min at 4°C. Equal amounts of total protein lysates (15 µg) were resolved by SDS-PAGE, transferred onto PVDF membranes and probed with antibodies. c-Cbl (C-15) antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). pS621c-Raf antibody was from Pierce Thermo Scientific (Lafayette, CO). Lyn, Fgr, pY416-SFK, AhR, Vav1, Slp76, p47^phox, c-Raf, pS259c-Raf, pS289/296/301c-Raf, VDR, RARα, GAPDH, horseradish peroxidase anti-mouse and horseradish peroxidase anti-rabbit were from Cell Signaling (Danvers, MA, USA). Enhanced chemiluminescence ECL reagent (GE Healthcare, Pittsburg, PA) was used for detection.