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. 2010 Jan 11;285(10):6952–6959. doi: 10.1074/jbc.M109.055731

FIGURE 3.

FIGURE 3.

Enhanced ubiquitination of EGFR in DGKδ-deficient cells is not due to an effect on c-Cbl. A, HeLa cells were transfected with scrambled or DGKδ siRNA duplexes. In the left panel, cells were starved for 16 h and then treated with EGF (10 ng/ml) for 5 min. In the right panel (steady-state), cells were grown in medium with 10% serum for 48 h. EGFR was immunoprecipitated from cell lysates (1 mg of protein for EGF-treated cells and 1.5 mg of protein for steady-state cells) and then ubiquitin (Ub), EGFR, and DGKδ were detected by Western blotting the immunoprecipitation or cell lysates. Ub band densities (arbitrary units) are shown above the blot. B, HeLa cells were transfected with Myc-tagged wild-type or Y1045F EGFR and then DGKδ was knocked down using siRNA. After 48 h EGFR (anti-Myc), DGKδ, and actin were detected by immunoblotting. EGFR band densities (arbitrary units) are shown above the blot. C, HeLa cells were transfected with scrambled or DGKδ siRNA duplexes, and then grown in complete medium or starved for 24 h. DGKδ, EGFR, transferrin receptor (TfnR), or actin were then detected in cell lysates by Western blotting. TfnR and EGFR band densities (arbitrary units) are shown above the blots. D, HeLa cells were transfected with scrambled or DGKδ siRNA duplexes, starved for 16 h, and then treated with EGF (5 ng/ml) for 0–30 min. Total EGFR, pY1045EGFR, actin, and DGKδ were detected in cell lysates by Western blotting. Band densities (arbitrary units) of pY1045EGFR normalized to total EGFR are shown above the blot. E, HeLa cells were transfected with V5-tagged c-Cbl and scrambled or DGKδ siRNA duplexes. After starvation for 16 h, the cells were exposed to 50 or 100 ng/ml EGF for 10 min, and then c-Cbl was immunoprecipitated from 1 mg of cell lysates. EGFR, c-Cbl (anti-V5), and DGKδ were detected in the precipitates and cell lysates by Western blotting. F, HeLa cells were transfected with scrambled or DGKδ siRNA duplexes, starved for 16 h, and then treated with EGF (5 ng/ml) for 0–30 min. Total c-Cbl, pY774-c-Cbl, and DGKδ were detected in cell lysates by Western blotting. All blots are representative of at least three independent experiments.