Figure 5.

RAB21 KO affects the subcellular localization of retromer and WASH and endosome morphology. (A) GFP-VPS35 was stably expressed in VPS35 KO HeLa cells, and two RAB21 KO cell pools were constructed on this basis. Endogenous VPS29, VPS26A, SNX27, WASHC4, ANKRD27 and RAB21 were coprecipitated by GFP-VPS35 in GFP-Trap assays. (B) WT and RAB21 KO HeLa cells were immunostained for VPS35. (C-D) HA-SNX27-expressing WT and RAB21 KO HeLa cells were immunostained for VPS35 along with HA. The Pearson’s coefficients (n > 25 cells from three independent experiments) are shown in D. (E-F) WT and RAB21 KO HeLa cells were immunostained for VPS35 along with WASHC2A. The Pearson’s coefficients (n > 25 cells from three independent experiments) are shown in F. (G-H) WT and RAB21 KO HeLa cells were immunostained for SNX27 along with EEA1. The Pearson’s coefficients (n > 25 cells from three independent experiments) are shown in H. (I-K) RAB21 KO and RAB21 KO re-expressing GFP-RAB21 HeLa cells were mixed and seeded onto coverslips for VPS35 or SNX27 immunostaining. The Pearson’s coefficients (n > 20 cells from three independent experiments) between VPS35 or SNX27 and RAB21 are shown in K. (L-M) GFP-2×FYVE was expressed in WT and RAB21 KO HeLa cells and was observed for endosome morphology. The percentage of malformed endosomes with enlarged tubules was analyzed, and the quantitative results are shown in M (n > 30 cells from three independent experiments). Scale bars: 10 μm. The graphs express the mean ± SEM. **P <0.01; NS, not significant.