Ps259c raf

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.

Whole cell lysates of HL-60 cells were prepared using 200 μL of a Pierce M-PER lysis buffer solution (Pierce, Rockford, IL) containing phosphatase and protease inhibitors (Sigma, St. Louis, MO). Lysates were centrifuged at 16,000 RCF for 30 min. Protein lysates were resolved by gel electrophoresis and transferred onto Immobilon-P PVDF membrane (Millipore Corporation, Billerica MA). Equal amounts of protein lysates (25 μg) were resolved by SDS-PAGE gel electrophoresis. For immunoprecipitation, equal amounts of protein (250 μg) were prECLeared with 30 μL of A/G protein beads (Santa Cruz Biotechnology, Santa Cruz, CA). Lysates were then treated 1/100 volume of antibody and 50 μL of beads overnight at 4 °C prior to electrophoresis. Blots were probed with antibodies: AhR (H211), c-Cbl (C-15)- (Santa Cruz Biotechnology, Santa Cruz, CA); CD38 (BD Biosciences, San Jose, CA); phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (D13.14.4E), p44/42 MAPK (ERK1/2) (137F5), pS221 MEK1/2, MEK1/2, Lyn, Fgr, Vav1, p47^phox, c-Raf, pS259c-Raf, GAPDH (Cell Signaling, Danvers, MA). Horseradish peroxidase anti-mouse and anti-rabbit antibodies (Cell Signaling, Danvers, MA) and ECL (GE Healthcare, Pittsburgh, PA) were used during detection. Blots were repeated at least three times.