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. Author manuscript; available in PMC: 2011 Nov 10.
Published in final edited form as: Structure. 2010 Nov 10;18(11):1536–1547. doi: 10.1016/j.str.2010.08.010

Figure 6. A function PTAP binding site in TSG101 is not needed for EGFR downregulation.

Figure 6

(A) HeLa cells were starved in serum-free medium for 1 h at 37°C and then incubated with EGF (50 or 100 ng/ml) for 1 h at 37°C. Protein extracts were analyzed by immunoblotting with a polyclonal antibody to EGFR (RK-2) and a monoclonal antibody to TSG101. (B) After a 72-h treatment with TSG101 RNAi, cells were starved in serum-free medium for 1 h at 37°C and then incubated with 50 ng/ml EGF for 1 h at 37°C. Mock and TSG101KD extracts were analyzed by immunoblotting for EGFR and TSG101. (C) After a 48-h treatment with TSG101 RNAi, cells were transfected with TSG101 constructs (rescue). At 20 h post-transfection, cells were starved in serum-free medium for 1 h at 37°C and then incubated with 50 ng/ml EGF for 1 h at 37°C. Extracts were analyzed by immunoblotting for EGFR and GFP-TSG101 with a polyclonal antibody to GFP. Transfection efficiency was higher than 50% in each experiment confirmed by fluorescence microscopy visualizing cells expressing GFP-TSG101. (D) Levels of EGFR were quantitated by x-ray film densitometry and percentages of undegraded EGFR upon EGF induction were plotted. Bars represent the mean ± SD from three independent experiments.