Fig. 5.

AMC is the sorting endosome. a Cells transfected with Myc-SNX1 and Myc-SNX3 were subjected to immunofluorescence (kinetics). Cells were incubated with anti-TfnR mouse monoclonal antibody for 1 h on ice. Thereafter, receptor–antibody complexes were allowed to be endocytosed and accumulated in AMC in the presence of 10 mM aspirin for 2 h. Cells were fixed and stained using polyclonal anti-Myc antibodies, followed by secondary antibodies. The colocalizations of internalized TfnR with SNXs are indicated by Pearson’s correlation (r). r average of values from five frames per coverslip and a representative from two independent experiments. SNX1-overexpressed cells treated with and without aspirin, r = 0.07 and r = 0.13, respectively. SNX3-overexpressed cells treated with and without aspirin, r = 0.26 and r = 0.36 respectively. b Similar to a, cells transfected with pDsred-SNX3 (top panel) and pDmyc-SNX3 (bottom panel) were subjected to immunofluorescence (kinetics). Arrows eTfnR clumped at perinuclear region. Image of fluorescence microscope, 100×. c A-431 (a) and HeLa (b) cells transfected with either control (Scr) or SNX3-specific siRNA duplexes for 36 h, were serum starved for 12 h prior to being either untreated (control) or treated with 100 nM and 40 nM EGF, respectively, in the presence of 10 μg/ml cycloheximide. Cells were lysed at the indicated time points and subjected to WB. Total EGFR for each sample was normalized to zero time point. Points average of normalized values from three independent experiments; bars ±SD. Results with significant statistical difference from control sample, *p < 0.05. d Cells treated with varying concentrations of aspirin for 4 h were subjected to cellular fractionation. The lysate fractions were then subjected to WB using indicated antibodies. Numerical ratio of membrane to cytosol