Genomic deletion of VPS35 does not cause retrograde sorting defects of CI-MPR. (A) Western blot of lysates from clonal HeLa cell lines with a disruption of the VPS35 gene in exon 5 or exon 8 (left). Immunofluorescent staining of endogenous GLUT1 (red) and endogenous LAMP1 (green) in HeLa cells and in two distinct VPS35-KO cell lines (right). (B) Staining of endogenous CI-MPR (red) and TGN46 (green) in parental HeLa cells and two VPS35-KO cell lines. The colocalization (coloc) between the TGN marker TGN46 and CI-MPR was quantified over three independent experiments. (C) Uptake assay with an antibody against the extracellular domain of CI-MPR in HeLa and VPS35-KO cell lines. Delivery of the CI-MPR–antibody complex (red) to the TGN46 (green)-labeled TGN was quantified after 30 and 60 min of uptake at 37°C over three independent experiments. (D) CI-MPR degradation assays in parental HeLa and VPS35-KO cells treated with the ribosomal inhibitor cycloheximide for indicated time points (left). Graph shows the degradation kinetics averaged over four independent experiments. Immunofluorescence of endogenous CI-MPR (red) and endogenous LAMP1 (green) in HeLa and VPS35-KO cell lines quantified over three independent experiments (right). Bars, 10 µm. Error bars indicate SD. *, P < 0.05 compared with control conditions in a t test.