Fig. 7. Comparison of the distribution of SNX4 and amphiphysin 2 with endocytosed transferrin receptor. (A) HeLa cells were transfected with full length SNX4 in pRK5myc. Before PFA fixation, cells were starved and incubated with Alexa 488-transferrin for 15 minutes at 37°C. Then, they were stained with anti-myc antibody followed by Cy3-coupled anti-mouse antibody. (B) HeLa cells were co-transfected with SNX4 in pRK5myc and Bramp2 in pRK5. Before PFA fixation, they were starved and incubated with Alexa 488-transferrin for 15 minutes at 37°C. Then, they were stained with anti-myc and anti-BIN1 antibodies, followed by Cy3 and Cy5 conjugated secondary antibodies. Images were acquired on a confocal microscope and pseudo-colored with Metamorph. Co-localization of the 3 fluorochromes appears in white.