The CI-MPR accumulates in VPS35-positive sorting endosomes but does not localize to the VPS35-decorated subdomain. (A) Immunofluorescent staining of endogenous CI-MPR (red) and endogenous EEA1 (green) in HeLa cells and clonal VPS35, SNX1/2, and SNX5/6 double-KO cell lines. The colocalization was quantified over three independent experiments. (B) Immunofluorescent staining of endogenous CI-MPR (red) and endogenous VPS35 (green) in HeLa cells and clonal SNX1/2 and SNX5/6 double-KO cell lines. The colocalization was quantified over three independent experiments. (C) Costaining of indicated endogenous proteins in WT HeLa cells and analysis of colocalization by conventional confocal microscopy and STED superresolution microscopy. The colocalization between CI-MPR and VPS35 and CI-MPR and SNX1 was quantified across 12 images acquired in two independent experiments with conventional confocal microscopy. Bars: (main images) 10 µm; (insets) 3 µm. Error bars indicate SD. *, P < 0.05 compared with control conditions in a t test.