KO of the SNX-BAR proteins causes a complete loss of retrograde sorting of the CI-MPR in HeLa cells. (A) Immunofluorescent analysis of the endogenous CI-MPR (red) and endogenous TGN46 (green) in clonal VPS35 and SNX1/2 and SNX5/6 double-KO cells. The colocalization was quantified over three independent experiments. (B) Uptake assay with an antibody against the extracellular domain of the endogenous CI-MPR over 30 and 60 min of uptake at 37°C. Delivery of the antibody/receptor duplex to the TGN was analyzed through costaining of the internalized antibody (red) with endogenous TGN46 (green). The colocalization was quantified over three independent experiments. (C) GFP-tagged SNX5 and GFP-tagged SNX6 were lentivirally expressed in SNX5/6 double-KO cells lines, and localization of endogenous CI-MPR (red) and endogenous TGN46 (blue) was quantified over three independent experiments. Bars, 10 µm. Error bars indicate SD. *, P < 0.05 compared with control conditions in a t test.