Figure 6.
Endocytosis of EGFR, but not of TfR, is inhibited in cells where ubiquitination of the EGFR is inhibited because of overexpression of hSpry2. (A) HeLa cells transfected with HA-ubiquitin and with or without either GFP-tagged hSpry2 or d.n. Grb2 were incubated with 60 ng/ml EGF as indicated. The EGFR was immunoprecipitated, and the precipitate was subjected to Western blotting with antibody to HA. (B) HeLa cells on coverslips were transfected with GFP-tagged hSpry2 for 24 h. Rhodamine-EGF (15 ng/ml; top panel) or TexasRed-Tf (20 μg/ml; bottom panel) was added at 37°C for 15 min before the cells were fixed. Bar, 10 μm. (C) Mock-transfected HeLa cells (▪) or HeLa cells transiently overexpressing GFP-tagged hSpry2 (•) for 24 h were incubated with 125I-EGF (1 ng/ml) on ice for 15 min and chased at 37°C for the indicated time periods. Analysis of internalized EGF was performed as described in MATERIALS AND METHODS. The data represent one typical experiment with six parallels ± SD.