Cells were lysed with Non-ident NP40 buffer as previously described [25 (link)]. Xenograft tissues were lysed similarly, including a previous step of mechanical disruption. Total protein concentrations were determined with the Bio-Rad protein quantification reagent (Bio-Rad Laboratories, Inc.). Equal amounts of protein (30–50 μg) were resolved by SDS-PAGE on 7–12% gels under reducing conditions. Separated proteins were transferred onto PVDF membranes, blocked with 5% milk or BSA in TBS/T, and incubated overnight with the following primary antibodies: p-Y1234/Y1235 MET, p-Ser473 AKT, p-Thr202/Tyr204 ERK1/2, p-Ser235/236 S6, pan-AKT, and pan ERK (all from Cell Signaling Technology). Anti-β-actin antibody was obtained from Millipore Corporation. The anti-HA high affinity antibody (clone 3 F10) was purchased from Roche.
Membranes were incubated with appropriate secondary antibodies and signals were detected with the ECL kit (Amersham Pharmacia Biotech) or with infrared fluorescence on an Odyssey imager (Li-Cor biosciences).
Immunoprecipitation was performed using the Dynabeads® Protein G kit following manufacturer’s instructions (ThermoFisher Scientific, #10003D).
With the exception of samples from in vivo tumors and organotypic slices, immunoblots shown are representative of at least three independent experiments.
Membranes were incubated with appropriate secondary antibodies and signals were detected with the ECL kit (Amersham Pharmacia Biotech) or with infrared fluorescence on an Odyssey imager (Li-Cor biosciences).
Immunoprecipitation was performed using the Dynabeads® Protein G kit following manufacturer’s instructions (ThermoFisher Scientific, #10003D).
With the exception of samples from in vivo tumors and organotypic slices, immunoblots shown are representative of at least three independent experiments.