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Fig. 5. Recruitment of the 5-HT_4(a)R by SNX27a to early endosomes in A431 cells. (A) A431 cells transiently transfected using EFFECTENE with the cMyc-tagged-5-HT_4(a)R together with HA-tagged SNX27a (a-d) or HA-SNX27a ${Delta}$ PDZ (e-h) and the cMyc-tagged-5-HT_4(a)R ${Delta}$ SCF receptor together with HA-SNX27a (i-l) were processed for indirect immunofluorescence using the polyclonal anti-Myc antibody (visualized in green using goat anti-rabbit IgG conjugated to FITC) (a,e,i) and the monoclonal anti HA (visualized in red using goat anti-mouse IgG conjugated to AlexaFluor 555) (b,f,j). Merge images (c,g,k) were further magnified to show detail (d,h,l). Yellow staining highlights colocalization between 5-HT_4(a)R and SNX27a. (B) Cells transfected as described in A were processed for indirect immunofluorescence, initially using the polyclonal anti-Myc antibody visualized in green (a,e,i) and the monoclonal anti-EEA1 antibody (visualized in blue using goat anti-mouse IgG conjugated to AlexaFluor647) (b,f,j), followed by incubation with a biotin-labelled monoclonal anti-HA antibody (visualized in red using streptavidin conjugated to Texas Red) (a,e,i). Overlaid images (c,g,k) were further magnified to show detail (d,h,l). Yellow staining highlights colocalization of the 5-HT_4(a)R and SNX27a, whereas purple indicates colocalization of SNX27a and EEA1, and cyan indicates colocalization of 5-HT_4(a)R and EEA1. White staining indicates colocalization between the 5-HT_4(a)R, SNX27a and EEA1. Bars, 10 µm.

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