Fig. 2. EGF-induced Eps15 phosphorylation depends on the ubiquitination function of c-Cbl and Y1045 of the EGFR. (A) CHO cells co-transfected with wild-type EGFR, FLAG-tagged Eps15 and empty vector, wild-type c-Cbl, or 70Z-Cbl were stimulated with EGF. Anti-FLAG immunoprecipitates (IP) were first probed with mAb to PY, stripped and reprobed with mAb to the FLAG tag. Anti-EGFR IPs were similarly probed in succession with anti-PY and anti-EGFR mAb and total cell lysates were immunoblotted (IB) with mAb to the HA-tag, as indicated. (B) CHO cells co-transfected with full-length c-Cbl, FLAG-tagged Eps15 and EGFR-WT or EGFRY1045F were stimulated with EGF. Anti-FLAG and anti-EGFR immunoprecipitates were immunoblotted with antibodies to PY (PY), FLAG-tag or EGFR.