Horseradish peroxidase anti rabbit

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.

The harvested cells were lysed in RIPA buffer containing protease and phosphatase inhibitors (Merck Millipore). The protein concentrations from the cell lysate, separate cytosol, and nuclear lysate were determined by a BCA Protein Assay Kit (Merck Millipore). Sixty micrograms of protein per well were subjected to SDS-PAGE. After electrophoresis, the proteins were transferred to a nitrocellulose membrane. The transferred membranes were blocked in 5% (w/v) nonfat dry milk or 5% (w/v) BSA in TBS (0.5 M NaCl, 20 mM Tris-HCl, pH 7.4) with 0.1% (v/v) Tween 20 and probed for the first antibody, followed by incubation with a secondary antibody conjugated with horseradish peroxidase (anti-rabbit, Cell Signaling; anti-mouse, Cell Signaling, Danvers, MA, USA) with visualization by ECL (Merck Millipore) with photographic film development. The first antibodies used in this study were anti-GAPDH (Cell Signaling, #5174), anti-Bim (Cell Signaling, #2933), anti-Bcl2 (Santa Cruz Biotechnology, sc-7382), anti-Bcl-xl (Cell Signaling, #2762), anti-caspase-3(a) (Cell Signaling, #9661), anti-PARP (Cell Signaling, #9542), anti-p21 (Cell Signaling, #2947), anti-Puma (Cell Signaling, #4976), and anti-p53 (Santa Cruz Biotechnology, sc-126). Immunoblot images were quantitated by Image Studio Lite (LI-COR Biosciences, Lincoln, NE, USA).