Subcellular localization of HA-TGN38 in RBL-Syt IX⁻ cells. RBL-Syt IX⁻ (A-D) or control RBL cells (E) were transiently transfected with HA-TGN38 cDNA or cotransfected with both HA-TGN38 and Rab 11-GFP (B). Cells were grown on glass coverslips for 48 hours and either allowed to internalize Texas red-conjugated Tfn (50 μg/mL) for 5 minutes (A) or 30 minutes (C) prior to their processing for immunofluorescence or left untreated (B) or incubated with serotonin (200 μM) for the last 24 hours of growth to load the SGs (D-E). Cells were subsequently labeled with mouse anti-HA antibodies (A-E) followed by Cy3-conjugated anti–mouse IgG or double-labeled with mouse antiserotonin and rabbit anti-HA antibodies, followed by FITC-conjugated donkey anti–mouse and Cy3 anti–rabbit IgG (D-E). Bars represent 3 μm (A,D-E) and 5 μm (B-C). Arrows indicate colocalization between HA-TGN38 and serotonin.