Defects in retrograde membrane traffic to the TGN in SHIP164 knockout cells. (A) Western blot (in kD) of control and edited RPE1 cell clones for SHIP164 and GAPDH as a loading control. (B) Left: Fluorescence images of a parental control (left) and SHIP164 KO cells (right) immunolabeled with antibodies against EEA1 (green) and GM130 (magenta). Scale bar, 20 µm. Right: Quantification of large EEA1 endosomes (>1 µm²) per cell in control and SHIP164 KO cell clones. (C) Live fluorescence (inverted grays) image of parental control (top) and SHIP164 KO (bottom) cells expressing the endosomal marker GFP-2xHrs^FYVE. Scale bar, 20 µm. (D–F) Left: Fluorescence images of parental control (left) and SHIP164 KO cells (right) immunolabeled with antibodies against indicated protein (green) and GM130 (magenta). Scale bar, 5 µm. Right: Quantification of scattered cytoplasmic spots–to–Golgi complex ratio of indicated protein signal per cell in control and SHIP164 KO cell clones. (G) Left: Fluorescence images of SHIP164 KO cells expressing RFP alone (left) or both RFP and SHIP164 (right) immunolabeled with antibodies against TGN46 (green) and GM130 (magenta). Scale bar, 5 µm. Right: Quantification of scattered cytoplasmic spots–to–Golgi complex ratio of TGN46 signal per cell in SHIP164 KO cell clones expressing RFP alone (solid circles) or both RFP and SHIP164 (open circles). Data of B and D–F reflect three biological replicates and of G, two biological replicates. For B, D–F, and G: middle line, mean; bars, SD. **, P < 0.01; *, P < 0.5. Source data are available for this figure: SourceData F6.