Figure 2.

Knockdown of c-Cbl reduces EGF-dependent ubiquitylation of the EGFR in corneal epithelial cells. Stable hTCEpi cells lines encoding for tetracycline-regulatable c-Cbl-specific shRNA and DsRed (hTCEpi[-c-Cbl]) were generated as described in Materials and Methods. Human telomerase-immortalized corneal epithelial cells were treated without (−Dox) or with (+Dox) 2 μg/mL of doxycycline in growth media for 72 hours. (A) Cell images were collected using a Nikon Ti-E microscope under Brightfield and CY5 settings. Identical settings were used for both treated and untreated cells. (B) Varying amounts cell lysates (20 μg, 10 μg, and 5 μg decreasing concentrations are represented by the thickness of the black triangle) from hTCEpi (-c-Cbl) cells treated without (−Dox) or with (+Dox) 1 μg/mL of doxycycline for 72 hours were immunoblotted for either c-Cbl or α-tubulin (α-Tub) as indicated. The 200-kDa and 45-kDa molecular weight standards are indicated on the left of the immunoblots. (C) Human telomerase-immortalized corneal epithelial cells were treated without (−) or with (+) doxycycline and/or 100 ng/mL EGF as indicated. Cell lysates were prepared and immunoprecipitated for the EGFR. The immunoprecipitate was resolved for 7.5% SDS-PAGE and immunoblotted for Ub, pY99, or the EGFR. A portion of the total cell lysate was resolved on a gel and immunoblotted for c-Cbl. Molecular weight standards are indicated on the left of the blot. Shown is a representative experiment repeated at least three times. (D) Densitometric analysis of three immunoblots from the experiment performed in (C). Data are plotted as the ratio of ubiquitylated EGFR to total EGFR (top), phosphorylated EGFR to total EGFR (middle), or c-Cbl (average ± SEM; n = 3). Data were analyzed with a paired Student's t-test. *P < 0.05; **P < 0.01.