Total met

Tumor xenografts were excised and rapidly frozen in OCT compound (Tissue-Tek, Torrance, CA) and tumor tissue sections were cut on a cryostat (Leica, Buffalo Grove, IL). Following standard protocols, the sections were processed for immunolabeling with anti-CD31 (1:50 dilution) (Abcam, Cambridge, MA), anti-phospho-c-Met (1:300 dilution), total-Met (1:300 dilution) (Cell Signaling, Danvers, MA) or anti-HO-1 (1:50 dilution) (Novus Biologicals, Littleton, CO); and subsequently labelled with a species-specific horseradish peroxidase-conjugateed secondary antibody. In tissue sections stained with anti-CD31 antibody, mean vessel density was calculated by standard grid counting method at x400 magnification.

Freshly harvested cancer cells or xenograft tumors were lysed with 1X cell lysis buffer (Cell Signaling Technologies, Danvers, MA) containing protease and phosphatase inhibitors (Roche, Indianapolis, IN). Subsequently, 30–40 μg of protein was run on 4–12% Bis-Tris gels (Thermo Fisher, Waltham, MA), and protein was transferred onto nitrocellulose membranes (Thermo Fisher). Membranes were blocked for one hour using Licor Odyssey Blocking Buffer (Lincoln, NE) before immunoblotting using the following antibodies (all from Cell Signaling Technology, Danvers, MA, USA): pEGFR (#3777), total EGFR (#2239), pMet (#3077), total Met (#3127), pAKT (#4060), total AKT (#9272), pERK (#9101), total ERK (#4695), pS6 (#2211), and total S6 (#2317). Western blots were processed using Licor Odyssey Imaging System, and densitometry was performed using Licor Odyssey Imaging System software.

Cells were harvested and disrupted in a radioimmunoprecipitation assay lysis buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 0.1% SDS, 1% NP-40, 0.25% Sodium deoxycholate and 1 mM EDTA) with protease inhibitor cocktail, 1 mM NaF and 1 mM Na₃VO₄. Equal amounts (40 μg) of whole cell lysates were separated on an 8% SDS-PAGE gel. After electrophoresis, samples were electrotransferred to a nitrocellulose membrane (BioRad) and probed with the following primary antibodies at 4° C overnight: Phospho-c-Met (Y1234/Y1235) (Cell Signaling #3077), total Met (Cell Signaling #3127), and β-actin (Sigma #1978). After primary antibody incubation, the membranes were incubated with the species-appropriate horseradish peroxidase conjugated secondary antibody (Jackson ImmunoResearch), and detected with an enhanced chemiluminescence substrate (GE Healthcare and Perkin Elmer).