TALEN-mediated deletion of VPS35 in U2OS and knockdown of VPS35 in HeLa cells does not cause retrograde sorting defects of CI-MPR. (A) Western blot analysis of clonal U2OS osteosarcoma cells with a TALEN-mediated disruption of exon 4 of the VPS35 gene. The graph displays the level of endogenous CI-MPR/GAPDH quantified over three independent experiments. (B) Immunofluorescent staining of endogenous CI-MPR and endogenous TGN46 in human U2OS, with colocalization quantified over three independent experiments. (C) CI-MPR degradation experiment in U2OS cells treated with the ribosome inhibitor cycloheximide for the indicated periods. The abundance of CI-MPR was adjusted by the GAPDH signal and quantified over three independent experiments. (D) Immunofluorescent staining of endogenous GLUT1 (red) and endogenous LAMP1 (green) in HeLa cells treated with scrambled and VPS35-specific siRNA. The colocalization between LAMP1 and GLUT1 was quantified across 12 images acquired in two independent experiments. The knockdown (KD) efficiency was verified by Western blotting. (E) Immunofluorescent staining of endogenous CI-MPR (red) with the lysosomal marker LAMP1 (blue) and the TGN marker TGN46 (green). The colocalization of CI-MPR with the two markers was quantified over three independent experiments. Bars, 10 µm. Error bars indicate SD. *, P < 0.05 compared with control conditions in a t test.