Fig 1: Only double knockout of c-Cbl and Cbl-b significantly alters c-Met phosphorylation and detectable ubiquitylated c-Met.A, lysates were prepared from hTCEpi, hTCEpi Cas9, hTCEpi -c-Cbl, hTCEpi -Cbl-b, and hTCEpi -c-Cbl/-Cbl-b (DKO) cells. Lysates (20 μg) were resolved by SDS-PAGE, transferred to nitrocellulose, and immunoblotted using antibodies against total c-Met, c-Cbl, Cbl-b, and α-tubulin as a loading control. Shown are representative blots from an experiment repeated three times. B, lysates (20 μg) were prepared from hTCEpi Cas9, -c-Cbl, -Cbl-b, and DKO cells that were treated without (−) or with (+) HGF (50 ng/ml) for 30 min. Ubiquitylated proteins were immunoprecipitated using an antiubiquitin antibody. Immunoprecipitates (upper panels) were resolved by SDS-PAGE, transferred to nitrocellulose, and immunoblotted using antibodies against total c-Met. Whole cell lysates (WCLs) (lower panel) were probed for total c-Met and phosphorylated c-Met to ensure c-Met activation with HGF treatment. Shown are representative blots from an experiment repeated three times. C, immunoprecipitated c-Met densitometry was normalized to WCL c-Met densitometry. Graphed are average ± SD and individual data points. Data were analyzed via two-way ANOVA. D–F, Cas9 cells were pretreated with MG-132 and treated with or without HGF as indicated in methods. A portion of the WCL was kept for running separately. Lysates were either incubated with FK2 antiubiquitin (catalog no.: ST1200; MilliporeSigma) antibody or mouse immunoglobulin G (catalog no.: 5415; Cell Signaling Tech). Immunoprecipitated proteins were isolated using A/G Agarose beads as described in Experimental procedures section. Portions of the WCL (D) and immunoprecipitates (E and F) were resolved through SDS-PAGE and immunoblotted for ubiquitin (catalog no.: 8017; Santa Cruz), c-Met, and phosphorylated c-Met pY1234/1235 to ensure receptor activation. Our data indicate a ubiquitin-specific pulldown and are consistent with Schwertman et al. (58). G, hTCEpi Cas9, -c-Cbl, -Cbl-b, and DKO cells were treated with HGF (50 ng/ml) and harvested at the indicated time points (0–4 h). Lysates were resolved, and membranes were immunoblotted for total c-Met, phosphorylated c-Met (pY1234/1235), c-Cbl, Cbl-b, and α-tubulin. Shown are representative blots from an experiment repeated three times. H, phosphorylated c-Met immunoreactive bands in (D) were quantified and normalized to α-tubulin. Graphed are average ± SD and individual data points. Data were analyzed via a two-way ANOVA. p Values on (E) indicate statistical significance between Cas9 (black), -c-Cbl (blue), or -Cbl-b (green) and DKO cells. DKO, double KO; HGF, hepatocyte growth factor; HTCEpi, human telomerase–immortalized corneal epithelial cell.