Figure 8. Endocytosis of endogenous human K2P9.1. All images are confocal whole cell projections. K2P9.1 is shown in green. Scale bar: 10 μm. Endocytosis panels: A549 cells were surface-biotinylated on ice, warmed to 37 °C for 60 min, then stripped to remove external biotin and fixed. Cells were stained with anti-K2P9.1 and streptavidin-DyLight 650 (blue) to reveal internal biotin. Merge: superimposed K2P9.1 and biotin. Vesicles: 0.6 μm and 1.2 μm spots of K2P9.1 (green) and biotin (blue) detected by Imaris software. Coloc vesicles: colocalized 0.6 μm and 1.2 μm K2P9.1 and biotin spots. EEA1 panels: fixed A549 cells were stained with anti-K2P9.1 and anti-EEA1 (red). Merge: superimposed K2P9.1 and EEA1. Vesicles: 0.6 μm and 1.2 μm spots of K2P9.1 (green) and EEA1 (red) detected by Imaris software. Coloc vesicles: colocalized 0.6 μm and 1.2 μm K2P9.1 and EEA1 spots. Rab7 panels: as for EEA1 panels, except anti-Rab7 (red) as the counterstain. Rab11 panels: fixed A549 cells were stained with anti-K2P9.1 and anti-Rab11 (red). Merge: superimposed K2P9.1 and Rab11. Vesicles: 0.6 μm spots of K2P9.1 (green) and Rab11 (red) were detected by Imaris software. Coloc vesicles: colocalized 0.6 μm spots of K2P9.1 and Rab11.