Figure 5
SNX5 is recruited to regions of the plasma membrane rich in PI(3,4)P 2 . (A) HEK-GFP-SNX5 cells were transfected with EGF-R and 24 h later serum-starved overnight and either left untreated or treated with 100 ng/ml EGF for 3 min at 37°C, as indicated. Cells were fixed and stained with anti-human EGF-R antibody followed by Alexa568-conjugated goat anti-mouse IgG for detection of surface EGF-R. Optical sections were taken to highlight the plasma membrane. Arrows indicate membrane ruffles showing co-localization of GFP-SNX5 and EGF-R (B) HEK-GFP-SNX5 cells were transfected with mCherry-TAPP1-PH and 24 h later serum-starved overnight then either left untreated or treated with 100 ng/ml EGF for 5 min at 37°C and then fixed. Arrows indicate co-localization of GFP-SNX5 and mCherry-TAPP1-PH (C) HEK-GFP-SNX5 cells were serum-starved overnight and either left untreated or treated with 100 ng/ml EGF for 3 min at 37°C, in the presence or absence of 100 nM AG1478, as indicated, and fixed. Boxed images are shown magnified on upper right hand corner. (D). Cells from (C) were scored for plasma membrane GFP-SNX5 by epifluorescence microscopy. 100 cells in triplicate were analysed. Shown is mean and error bars represent standard deviation. *** p <0.005. Bars = 10 μm.