KO of VPS29 does not result in loss of CI-MPR or CI-MPR dispersal from the TGN. (A) Western blot analysis of indicated proteins in parental HeLa cells and four VPS29-deficient cell lines generated with CRISPR-Cas9. (B) Immunofluorescence of endogenous GLUT1 (green) and endogenous LAMP2 (red) in HeLa cells and a VPS29-KO cell line. The colocalization was quantified across 16 images taken from two independent experiments. (C) Lentiviral reexpression of VPS29-myc fully restores GLUT1 localization at the plasma membrane. The colocalization was quantified across 12 images acquired in two independent experiments (D) Immunofluorescent staining of endogenous CI-MPR (red) and the endogenous TGN marker TGN46 (green) in parental HeLa and VPS29-KO cell lines. The colocalization was quantified across 12 images acquired in two independent experiments. Bars, 10 µm. Error bars indicate SD. *, P < 0.05 compared with control conditions in a t test.