Figure S5.

PTPN14 colocalizes with endosomes, but pY374-PKCδ level does not affect total RAB protein expression. (a) Colocalization of PTPN14 with the indicated RABs determined by PLA. Cells were stimulated with 20 ng/ml EGF for 15 min, fixed, and stained with anti-rabbit PTPN14 and anti-mouse RAB5, RAB7, or RAB11, followed by proximity ligation using anti-rabbit PLUS and anti-mouse MINUS probes. (b) Electron micrographs of immunogold-labeled PTPN14 in various intracellular organelles as indicated. Immuno-EM localization of FLAG-tagged PTPN14. MDA-MB-231 cells were incubated with dox to induce expression of FLAG-tagged PTPN14 and were then fixed and processed for immuno-EM. Labeling was evident on putative early endosomal compartments (E) and associated membranes (arrows) as well as late endosomal compartments (L). Mitochondria (M) showed variable labeling. N, nucleus; PM, plasma membrane; G, Golgi. (c and d) Representative Western blot (c) and quantitation relative to total protein (d) from n = 3 independent experiments showing RAB5, RAB7, and RAB11 protein levels in pCas9Vec and PTPN14-KO cells stimulated with EGF for the indicated times.