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Fig. 1. Ubiquitin ligase activity of c-Cbl contributes to clathrin-mediated EGFR internalization. (A) Wild-type c-Cbl and 70Z-Cbl proteins used in this study. 70Z-Cbl lacks 17 amino acids including part of the Ring finger domain. (B) CHO cells were transfected with empty vector, c-Cbl or 70Z-Cbl. Western blots of total cell lysates were probed with antibodies against c-Cbl. (C) CHO cells were co-transfected with EGFR, HA-ubiquitin and empty vector, c-Cbl or 70Z-Cbl. Anti-EGFR immunoprecipitates from EGF-stimulated cells were immunoblotted with anti-HA mAb to detect ubiquitinated EGFR and pAb to the EGFR to detect total EGFR protein amounts. (D) CHO cells co-expressing the EGFR and empty vector, c-Cbl or 70Z-Cbl were incubated at 37°C with 1 ng/ml ¹²⁵I-labelled EGF. Internalization was monitored as outlined in Materials and Methods. Presented is the ratio of internalized to total-cell-associated radioactivity. Expression controls of the Cbl proteins are shown in B. (E) CHO cells expressing wild-type EGFR (EGFR-WT) or EGFR-Y1045 in the presence or absence of exogenous c-Cbl were used in an internalization assay as described in D. (F) CHO cells were co-transfected with full length c-Cbl and wild-type EGFR and treated as described in D, using 20 ng/ml radiolabelled EGF (control), or subjected to hypotonic shock followed by incubation with ligand in the absence () or presence () of K⁺ ions. Experiments in D and E contained triplicate samples, error bars represent standard deviations. In F, both values of duplicate samples are shown and means are connected by a line.

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