Raf 1 c 20

Cells were collected in NP-40 lysis buffer (containing 1% NP-40, 20 mmol/L Tris-HCl (pH 8.0), 137 mmol/L NaCl, and 10% glycerol) with 1× protease/phosphatase inhibitor cocktail (Thermo)). Protein concentration was determined using the DC protein assay (Bio-rad). Protein (30 μg) was separated using an 8% PAGE-SDS gel, and transferred to Immobilon-P membranes (Millipore). Membranes were incubated overnight at 4°C with the indicated antibodies: FAK (BD Biosciences), p-Y925-FAK, ppERK1/2, ERK1/2, c-Src, p-Y416-SFK, SFK (Cell Signaling), Raf-1 (C-20), Raf-B (F-7) (Santa Cruz), β-actin (Sigma), or α-Tubulin (Calbiochem). For ECL detection, blots were incubated with secondary goat anti-rabbit or goat anti-mouse horseradish peroxidase–conjugated antibodies (GE Healthcare) and detected by enhanced chemiluminescence (ECL) (Pierce). For Odyssey CLx imaging blots were incubated with secondary goat anti-rabbit (IRDye 800CW) or goat anti-mouse (IRDye 680RD) (Li-Cor).
For immunoprecipitation (IP) assays, lysates were rotated with pre-clearing matrix F beads for 30 minutes at 4°C. Protein (500 μg) in 500 μl of lysis buffer was then incubated for 1 hour at 4°C with the indicated antibody (5 μg Raf-1 (C-20) or rabbit IgG (cell signaling)) prior to being incubated with 40 μl of the IP/WB Optima F beads (Santa Cruz) overnight at 4°C.