FIGURE 4:

ST-RFP is not retained in the Golgi in GARP-deficient RPE1 cells. (A) WT, VPS53-KO, and VPS54-KO RPE1 cells were co-transfected with plasmids encoding ST-RFP and LAMP2-GFP for 4 h, followed by staining for the Golgi marker GM130. Microscopic images in A show individual channels in grayscale on top of each merged image. Bottom panels in A are the line scan plots of relative intensity over distance that demonstrates the overlap between the channels. Line scan analysis was done using ImageJ. (B) WT, VPS53-KO and VPS54-KO RPE1 cells were co-transfected with plasmids encoding ST-RFP and LAMP2-GFP for 20 h, followed by staining for the Golgi marker GM130. Insets are zoomed in views of the small white-boxed areas (10× inset). Colocalization of ST-RFP with LAMP2-GFP was determined by calculation of the Pearson’s correlation coefficient (right panel) in approximately 20 cells. Statistical significance was calculated using one-way ANOVA. ****P ≤ 0.0001.