Fig. 2. Intracellular localization of human SNX5 PX domain in transfected HeLa cells. (A) HeLa cells were transiently transfected with GFP-SNX5_PX and after 12 hours of transfection, cells were fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton-X-100, and co-stained for EEA1, Lamp-1 and SNX1. EEA1 was detected with human anti-EEA1 antibodies followed by human anti-Ig Alexa568, and Lamp-1 and SNX1 were detected with mouse monoclonal antibodies and goat anti-mouse IgG-Alexa568 conjugate. (B) EEA1 was detected with human anti-EEA1 antibodies and FITC-goat anti-human IgG and SNX1 as for A. Control incubations demonstrated no cross-reactivity between the anti-Ig conjugates and the irrelevant primary antibody. Confocal images were collected with identical iris settings. Superimposed images (Merge) reveal regions of colocalization. Inserts show regions of the cell at higher magnification. Bar, 10 µm.