Loss of VAMP-3 affected transit of cargoes to endosomes. Washed platelets (1 × 109/mL) were incubated with Alexa 647-Fg and Oregon Green 488-Dextran at final concentrations of 1 µM each and incubated at 37°C for 30 to 60 minutes and prepared for immunofluorescence microscopy, as described in Methods. Platelets were incubated with anti-Rab4 rabbit polyclonal antibody (1:250 dilution) and anti-Rab11 rabbit polyclonal antibody (1:250 dilution), and then with Alexa 568-conjugated goat anti-rabbit IgG secondary antibody (1:1,000 dilution). Images were taken using the Nikon Ti-E N-STORM/N-SIM super-resolution microscope and digitally magnified by ×30. The Alexa 647-Fg signal was faux-colored blue to aid in identifying overlaps. Scale bars, 1 µm. Data are representative of 2 independent experiments. (C) Pearson’s correlation coefficients were calculated using the NIS-Elements v3.2 N-SIM/STORM suite software to show overlap among (i) Alexa 647-Fg and Oregon Green 488-Dextran (depicted in cyan), (ii) Alexa 568-Rab4 and Alexa 647-Fg (depicted in magenta), (iii) Alexa 568-Rab4 and Oregon Green 488-Dextran (depicted in yellow), (iv) Alexa 568-Rab11 and Alexa 647-Fg (depicted in magenta), and (v) Alexa 568-Rab11 and Oregon Green 488-Dextran (depicted in yellow), at the indicated times. Overlap of all 3 fluorophores is depicted in white in the merge panel. Graph shows the mean ± SEM of 2 independent experiments, with at least 30 cells per field, taken over 3 to 4 fields per time. Statistical analyses were performed using Student t test; *P ≤ .05; **P ≤ .01; ***P ≤ .001; n.s., not significant.
