FIGURE 7:

Hook2 and PCM1 are in a complex with Rab8a. (A) Lysates from HEK cells transfected with GFP::Rab8a or GFP::Rab10 were lysed and incubated with G protein–coated beads coupled to mouse anti-GFP antibodies and processed for WB analysis with rabbit antibodies anti-PCM1, anti-Hook2, and anti-GFP and immunoglobulin light chain–specific secondary antibodies. MW markers are indicated on the left in kilodaltons. Note that Hook2 and PCM1 were coimmunoprecipitated with GFP::Rab8a but not with GFP::Rab10. (B, C) Control or Hook2 siRNA (H1–3) transiently depleted cells were further transfected with GFP::Rab8a or GFP::Rab10 cDNA in addition to control or Hook2 siRNA. After 7 d, the cells were processed for WB analyses (B) with the mitochondrial marker prohibitin as a loading control. In parallel, the cells were fixed in PFA, permeabilized with Tx100, stained with antibodies raised against Hook2 and acetylated tubulin, and compared by immunofluorescence and confocal microscopy analysis (C). Bars, 10 μm; inset magnification, ×15. Arrowheads indicate GFP::Rab8a–positive cilia, even in the absence of Hook2 (siRNA Hook2 panels). (D) The number of transfected cells with a cilium in each siRNA condition is quantified and normalized to 100% for the control cells (n = 5). (E) Control or Hook2 siRNA (H1–3) transiently depleted cells were further transfected with GFP::Rab8a in addition to control or Hook2 siRNA as in B and C. After 7 d, the cells were processed for confocal microscopy analysis after immunofluorescence staining with antibodies raised against Hook2 and TGN46. Bars, 10 μm; inset magnification, ×15. Arrows indicate the TGN area of transfected cells with a cilium. (F) The volume of the TGN was measured in GFP::Rab8a–transfected cells in control and Hook2 siRNA conditions (n = 3, p < 0001). Note that GFP::Rab8a restored the ciliogenesis defect but not the Golgi compaction induced by Hook2 depletion. See also Supplemental Figures S5 and S6.