Fig. 9
ATP11C is downregulated by clathrin-mediated endocytosis upon PKC activation. HeLa cells stably expressing ATP11C-HA were transfected with non-targeting siRNA (siControl) (a, d) or siRNAs against clathrin heavy chain (CHC) (b, e) or PKCα (c, f). (see Supplementary Fig. 4). Each siRNA-treated sample was treated with vehicle alone (Mock), 400 nM PMA (PMA), or 1 μM A23187 in the presence of CaCl2 (A23187) for 15 min, followed by fixation. The fixed cells were permeabilized and incubated with anti-HA, anti-ATP1A1, and anti-CHC antibodies, followed by Cy3-conjugated anti-rat, Alexa Fluor 647-conjugated anti-rabbit, and Alexa Fluor 488-conjugated anti-mouse secondary antibodies. Scale bars, 20 μm. d–f The cells in which ATP11C localized to the plasma membrane (PM), to the plasma membrane and endosomes (PM + E), or to endosomes (E) were counted; counts were normalized against the total number of counted cells. In each sample, 643–783 cells were counted. Graphs display averages ± SD from three independent experiments
