“…Following lysis in RIPA bu er (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.5% DOC, 1% NP40, 0.1% SDS, 25 mM NaF, 2 mM Na 3 VO 4 , aprotinin, leupeptin, pefabloc each 10 mg/ml), protein concentrations were determined using DC Protein Assay (BioRad Laboratories, Hercules, CA, USA). Total cell protein (400 mg) was incubated with glutathione-Sepharose coupled peptides corresponding to the Ras binding domains (RBDs) (5 mg/ml beads) of RalGDS (Hofer et al, 1994) or Raf-1 (Kolch et al, 1996) for 1 h at 48C. The beads were washed three times with RIPA bu er, bound proteins eluted in sample bu er, heated for 5 min at 958C, separated on 12% SDS ± PAGE, transferred to nitrocellulose and subjected to immunoblotting with anti-Ras or anti-HA antibodies.…”