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. 2013 May 23;2(7):686–694. doi: 10.1242/bio.20134747

Fig. 6. The BBSome, Arl6 and Tubby are required for VPAC2 ciliary targeting.

Fig. 6.

(A,B) BBS2 and Tubby mRNA levels were reduced by more than 93% in rat thalamic cultures infected with lentivirus containing shRNAmiR against BBS2 (A) or Tubby (B) compared to cells infected with control lentivirus. mRNA levels were measured by RT-qPCR. Error bars represent the SD of triplicate PCR assays. ***P<0.001. GAPDH levels were used as an internal control for normalization. (C) Protein extracts from mouse thalamic cultures infected with control shRNA or Arl6 shRNAs were immunoblotted for Arl6 and actin (loading control). (D) Tubby and BBS2 are required for VPAC2 ciliary targeting. Rat thalamic cells were infected with lentivirus containing control shRNAmiR and shRNAmiRs against Tubby or BBS2 and immunostained for VPAC2 (green) and Arl13B (red). (E) Thalamic neuron culture cells were prepared and stained as in panel D. Approximately 500 cilia were counted and the percentage of VPAC2 positive cilia was plotted. (F) Knockdown of Arl6 affects VPAC2 ciliary targeting. Thalamic cultures were infected with control shRNA or Arl6 shRNAs or immunostained for VPAC2 (green) and Arl13B (red). (G) Arl6 is required for VPAC2 ciliary targeting. Rat thalamic neuron culture cells were prepared and stained as in panel F. Approximately 600 cilia were counted and the percentage of VPAC2 positive cilia was plotted. Error bars represent the SD among three different coverslips (E,G). ***P<0.001. DNA was labeled with DAPI <2?show=[to]?>(D,F). Insets show unmerged images of the region around a cilium highlighted with a white arrow. Scale bars: 5 µm.