Anti ras antibody

Cells were lysed either in SDS sample buffer (2% SDS, 125 mM DTT, 10% glycerol, 62.5 mM Tris-HCl, pH 6.8) or RIPA buffer (10 mM Phosphate buffer, 150 mM NaCl, 1% NP40, 0.1% sodium deoxycholate, and 0.1% SDS) containing protease inhibitors and phosphatase inhibitors. Equivalent amounts of proteins adjusted by cell number were loaded in each lane and separated on SDS- PAGE gels. Proteins transferred on PVDF membranes were probed with the following primary antibodies: antibodies specific for phospho-Erk (Thr 202/Tyr204), phospho-Mek1/2 (Ser 217/221), phosphor-cRaf (Ser 259), phospho-smad 2/3 (Ser 423/425) and Mek were from Cell Signaling Technology. Antibodies specific for RasGRP1 and Sos1 were from Santa Cruz Biotechnology (Dallas, TX, USA). The anti-Ras antibody was from BD Biosciences, the anti-Erk Ab was from Millipore (Billerica, MA, USA), the anti-Foxp3 Ab was from eBiosciences, and the anti-p actin anti-body was from Sigma (St. Louis, MO, USA).

Generation and characterization of the antiserum against the Gβ₁ subunit are detailed elsewhere [31 (link),50 (link)]. Specific antibodies against p87 and p101 were generous gifts from Michael Schaefer (Leipzig, Germany) and Len Stephens (Cambridge, U.K.), respectively. Monoclonal anti-p110γ antibody, mAb(A)_p110γ and mAb(B)_p110γ, were raised against full-length human p110γ using mouse hybridome cells and characterized earlier [37 (link)]. Large scale preparations of mAb(A)_p110γ were generated in cooperation with BioGenes, Berlin, Germany. mAb(B)_p110γ was described earlier [31 (link),40 (link),41 (link)]. Generation and characterization of monoclonal anti-p110γ antibody, mAb(C)_p110γ, raised against the N-terminal 210 amino acids of catalytic p110γ was detailed earlier [43 (link)]. Anti-Ras antibody was purchased from BD Biosciences (#610002). Anti-p110β antibody was purchased from Cell Signaling (#3011S). Proteins were fractionated by SDS/PAGE (10% acrylamide) and transferred to nitrocellulose membranes (Hybond™-C Extra, GE Healthcare). Visualization of specific antisera was performed using the ECL chemiluminescence system (GE Healthcare) or the SuperSignal^® West Pico Chemiluminescent Substrate (Pierce) according to the manufacturers’ instructions. Chemiluminescence signals were estimated using the VersaDoc™ 4000 MP imaging system (Bio-Rad).