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. 2014 Jan 20;204(2):215–229. doi: 10.1083/jcb.201304153

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© 2014 Kobayashi et al.

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Figure 6. — Rab8a functions downstream of Talpid3 and Cep290 in ciliogenesis. (A) Western blotting of Talpid3 and GFP after immunoprecipitation of RPE1 cell extracts stably expressing GFP-Rab8a or GFP-Rabin8 with antibodies indicated at the top of panel. IN, input. (B and D) RPE1 cells treated with control (siNS), Talpid3, or Cep290 siRNA were transiently transfected with plasmids expressing GFP and (B) Myc, Myc-Rab8a/QL, or Myc-Rab8a/TN, or (D) Flag, or Flag-Rabin8, and induced to quiescence for 48 h. Cells were processed for immunofluorescence with an anti-glutamylated tubulin (GT335) antibody. The percentages of GFP-positive RPE1 cells with primary cilia were determined. (C and E) RPE1 cells treated with control, Talpid3, or Cep290 siRNA were transiently transfected with plasmids expressing GFP-Centrin2 and (C) Myc, Myc-Rab8a/QL, or Myc-Rab8a/TN, or (E) Flag, or Flag-Rabin8, and induced to quiescence for 48 h. Cells were processed for immunofluorescence with an anti-PCM1 antibody. The percentages of GFP-Centrin2–positive RPE1 cells with concentrated PCM1 granules around centrosomes were determined. (B–E) Average of three independent experiments is shown. *, P < 0.01; **, P < 0.05.