Loss of the SNX1/2–SNX5/6 complex leads to an accumulation of CI-MPR in endosomes. (A) HeLa cells were transiently transfected with nontargeting siRNA, siRNA targeting VPS35 or an siRNA pool against SNX1, SNX2, SNX5, and SNX6. 72 h after transfection, endogenous protein levels were analyzed by Western blotting. (B) CI-MPR steady-state localization in retromer versus retromer-linked SNX-BAR–knockdown cells. Representative images (left) and blind scoring and quantification (right) of the percentage of cells displaying each phenotype in knockdown conditions. n = 3 independent experiments; nontargeting, 144 cells; SNX1+2+5+6, 169 cells; VPS35, 133 cells. (C) Immunofluorescence and colocalization analysis of endogenous CI-MPR and lysosomal marker LAMP1 and early endosomal marker EEA1 in SNX1, SNX2, SNX5, and SNX6 or VPS35-knockdown HeLa cells. (C, top right) n = 3 independent experiments; nontargeting, 130 cells; SNX1+2+5+6, 162 cells; VPS35, 103 cells. (C, bottom right) n = 3 independent experiments; nontargeting, 115 cells; SNX1+2+5+6, 131 cells; VPS35, 109 cells. (D) Immunofluorescence and colocalization analysis of endogenous CI-MPR and transition endosome markers SNX1 and VPS35 in SNX1, SNX2, SNX5, and SNX6 or VPS35-deficient HeLa cells. Bars: (main images) 20 µm; (insets) 5 µm. (D, top right) n = 3 independent experiments; nontargeting, 101 cells; SNX1+2+5+6, 100 cells. (D, bottom right) n = 3 independent experiments; nontargeting, 98 cells; VPS35, 119 cells (means ± SEM; two-way ANOVA compared with nontargeting control; *, P < 0.05; **, P < 0.01).