Anti nras sc 519

Cells were gently washed in PBS and then lysed using lysis buffer (1% NP-40, 50 mM Tris–HCl pH 7.5, 150 mM NaCl, complete protease inhibitor tablet, EDTA-free (Roche), 1 μM sodium orthovanadate, 1 mM sodium fluoride, and 0.1% β-mercaptoethanol). Lysed cells were scraped and transferred into a 1.5-ml microcentrifuge tube. Samples were incubated at 4°C for 20 min and then centrifuged for 15 min at 17,000x g at 4°C. Proteins (50 μg) were resolved on 10% SDS-polyacrylamide gels and transferred to nitrocellulose membranes (Bio-Rad). Alternatively, the cleared cell lysates were incubated with anti-Flag M2 affinity agarose beads (Sigma-Aldrich), overnight at 4°C. Then the beads were washed three times with the lysis buffer then resuspended with sample buffer and then resolved on 10% SDS-polyacrylamide gels and transferred to nitrocellulose membranes (Bio-Rad). Western blots were probed with the following antibodies: anti-Flag (M2) (Sigma-Aldrich), anti-c-NRAS (F155-277, Millipore), anti-NRAS (sc-519, Santa Cruz), anti-c-CBL (2747, Cell signaling), and anti-αtubulin (Millipore).