Monoclonal flag m2

Cell lysates were incubated with 10 ml of 50% slurry of protein-A/G sepharose beads for 1 h to preclear. One microgram of polyclonal MYC A14 or monoclonal FLAG M2 (Sigma) antibody was added to the supernatant for a minimum of 4 hours at 4°C, followed by a 1 hour incubation with 20 ml protein-A/G sepharose beads (Sigma). After four washes with lysis buffer, the precipitated proteins were analyzed by SDS-PAGE and transferred to PVDF membrane. Membranes were probed with the monoclonal FLAG M2 or monoclonal MYC9E10. Western blots were developed with the enhanced chemiluminescence (ECL) analysis system (GE Life Sciences).

Rabbit polyclonal antibodies against Src (#2109, 1:5000), phosphorylated (p)Src (#2101, 1:1000), pAKT (#9271, 1:1000), AKT (#9272, 1:1000), ERK (#9102, 1:1000), SYK (#2712, 1:1000), FAK (#3285, 1:1000), pTyr (P-Tyr-1000) (#8954, 1:2000), cleaved caspase-3 (#9664, 1:1000), cleaved PARP (#9541, 1:1000), PARP (#9542, 1:1000), cleaved caspase-9 (#9661, 1:1000), caspase-9 (#9508, 1:1000), and HA (#3724, 1:5000) were obtained from Cell Signaling Technologies. Polyclonal IgG (sc-2027), pERK (sc-7383, 1:500), CBL (sc-170, 1:500), SHP2 (sc-280, 1:1000), pMEK1/2 (sc-81503, 1:500), and MEK-1 (sc-6250, 1:500) were obtained from Santa Cruz Biotechnology. p-SHP2(Y542) (ab62322, 1:20,000) was obtained from Abcam. Monoclonal antibodies against Pan-Ras (OP40, 1:500), HA (12CA5, 1:500), and pTyr (4G10) (05–321, 1:1000) were obtained from Boehringer Ingelheim and Millipore, respectively. Monoclonal FLAG-M2 (F1804, 1:2000) and Vinculin (V9264, 1:2000) were obtained from Sigma.