FIGURE 5.
PMA treatment increases the co-localization of endocytosed KATP channels with the lysosomal marker Lamp1. A–F, effect of PKC activation on the intracellular distribution of endocytosed KATP channels. HEK293 cells expressing HA-Kir6.2 and SUR1 (A–D) plus Lamp1-green fluorescent protein (E and F) were allowed to internalize anti-HA antibodies at 37 °C for 1 h in the presence of either vehicle (DMSO, 0.1%) or PMA (100 nm). Following fixation, the cells were co-stained for HA-Kir6.2 (red) and the indicated marker proteins (green). EEA1 was for early endosomes, and the cation-independent mannose-6-phosphate receptor (M6PR) was for late endosomes. A, C, and E are representative of at least three independent experiments; the larger images in each panel represent a merger of the small images stained for HA-Kir6.2 (red) and marker proteins (green) at the bottom. The scale bars equal 10 μm. In B, D, and F, co-localization was quantified by particle analysis using the Image J software to generate Pearson correlation coefficients from at least 10 cells and from each of three independent experiments. The data shown are means ± S.E. * denotes significance at the p < 0.05 level assessed by Student's t test.