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Fig. 4. Effect of SNX16 on the EGF-induced degradation of EGFR. (A) COS-7 cells expressing FLAG-tagged SNX16^WT and SNX16^Y145A were treated with EGF for the indicated times and then immunoblotted with antibodies against EGFR, FLAG or actin. The activation of MAPK (phospho-ERK1/2) was also assessed by immunoblotting with anti-phospho ERK1/2 antibody. Anti-ERK1/2 antibody was used to detect levels of ERK1/2 as a loading control. (B) COS-7 cells were transfected with GFP-tagged SNX16 and incubated with Rhodamine-conjugated LysoTracker. Cells were treated with EGF for the indicated times. Fixed cells were processed to detect SNX16 (green) and LysoTracker (red) simultaneously by confocal microscopy. Bars, 10 µm. (C) COS-7 cells were treated with EGF at the indicated times. Cell surface proteins were biotinylated as described in Materials and Methods. Biotinylated proteins were recovered using Streptavidin and the amount of EGFR recovered was assessed by western blotting using anti-EGFR antibody.

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