Figure 4
Western blot analysis and esiRNA-mediated depletion of TRAPPC8. (A) Western blot analysis of whole cell lysate from RPE cells, probed with rabbit polyclonal TRAPPC8 antibody. Molecular mass markers are shown in kDa to the left. (B) Western blot analysis of lysates from RPE cells treated with TRAPPC8-specific esiRNA or mock-transfected control cells. Blots were probed with antibodies specific for TRAPPC8 or α-tubulin (loading control). (C) Quantification of cilia in RPE cells depleted for TRAPPC8 using TRAPPC8-specific esiRNA. The cells were fixed with PFA and stained with acetylated tubulin antibody for visualization of cilia. Three independent experiments were conducted with 100 cells counted per condition per experiment. P value (*) = 0.0227 using unpaired t test. (D) Selected immunofluorescence micrographs of GFP-Rabin8 expressing mock-transfected control cells or cells depleted for TRAPPC8. Cells were first treated with mock or TRAPPC8-specific esiRNA and then transfected with GFP-Rabin8 plasmid. Following serum starvation for 1 h, cells were fixed with PFA and stained with antibody against p150Glued to mark the centrosome (red). In Mock-transfected control cells, 92% of GFP-Rabin8 expressing cells showed GFP-Rabin8 at the centrosome whereas only 60% of the GFP-Rabin8 expressing TRAPPC8-depleted cells showed centrosomal GFP-Rabin8 localization (50 cells analyzed per condition).