Serum-starved cells were grown to confluence before treatment with human HGF (catalog no.: CYT-244; ProSpec). Ligand concentrations and treatment times are indicated in figure legends. Cells were subject to two washes of PBS in room temperature followed by equilibration to 4 °C for 5 min. Cells were harvested in radioimmunoprecipitation assay buffer (150 mM sodium chloride, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 10 mM sodium pyrophosphate, 100 mM sodium fluoride, 50 nM Tris [pH 8.0]) supplemented with protease inhibitor PMSF (2 mM; Calbiochem) and solubilized by end-over-end rotation for 10 min at 4 °C. Insoluble material was removed via centrifugation for 10 min at 4 °C. The resulting cell lysate was assessed by bicinchoninic acid assay (catalog no.: 23225; Thermo Scientific), diluted in SDS sample buffer, and boiled for 3 min prior to gel loading. Equal amounts of protein were loaded and resolved by 10% SDS-PAGE before transfer to nitrocellulose membrane. Membranes were immunoblotted using the indicated antibodies and the manufacturer’s instructions: total c-Met (catalog no.: 8198), phosphorylated c-Met pY1234/1235 (catalog no.: 3077), c-Cbl (catalog no.: 2747), Cbl-b (catalog no.: 9498), total p42/p44 MAPK (ERK1/2) (catalog no.: 4695) phosphorylated p42/p44 MAPK (ERK1/2) (catalog no.: 9101) (Cell Signaling Technologies), and α-tubulin (catalog no.: 6199) (MilliporeSigma). Following incubation with the appropriate horseradish peroxidase–conjugated secondary antibody (antimouse: catalog no.: 31450; Invitrogen; anti-rabbit: catalog no.: 7074; Cell Signaling Technologies), enhanced chemiluminescence was used to visualize immunoreactive bands in a Fotodyne imaging system. When comparing c-Met phosphorylation, samples were run on the same gel if possible (time courses, Figs. 2G and 3C). Otherwise, immunoblots were run, processed, and developed together (dose response, Fig. 3A). Each experiment was performed at least three independent times. Densitometry analysis was executed using the ImageJ software (National Institutes of Health). GraphPad/Prism 9 (GraphPad Software, Inc) was used for generating graphs and performing statistical analysis (57 , 62 (link), 75 (link)). Area under the curves were generated by plotting each individual experiment and finding the replicate areas before graphing in Prism.
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