Fig. 2.

Localization of AC5/6 and AKAP150 in renal primary cilia. (A) Kif3a^F/− cells were stained with antibodies that recognize both AC5 and AC6 (red, b) or AC6 only (red, e) and costained with antiacetylated tubulin antibody (green, a and d). The merged image shows colocalization in primary cilia (c and f). (Scale bars: 5 μm.) (B) IMCD3 cells were stably transfected with Flag-tagged AC5 or AC6 and stained with anti-Flag antibody (green). Flag-tagged AC5 (a and b) and AC6 (c and d) are localized in plasma membrane (a and c) and primary cilia (arrows in b and d). (Scale bars: a and c, 10 μm; b and d, 20 μm.) (C) Treatment with NKY80, a specific inhibitor of AC5, produces a dose-dependent decrease in CREB reporter activity in Kif3a^−/− cells but has no effect in Kif3a^F/− cells. Error bars indicate SD (n = 3). (D) Transfection of Kif3a^−/− cells with siRNA to AC5 reduces CREB reporter activity, whereas siRNA to AC6 or scrambled siRNA (control) have no effect. Expression of siRNAs does not affect CREB reporter activity in Kif3a^F/– cells (n = 3). (E) mIMCD3 cells were costained with anti-AKAP150 (red in b, e, and h) and antibodies (green) against acetylated tubulin (a), γ-tubulin (d), or α-tubulin (g). Merged images show localization of AKAP150 in primary cilia (c), centrosomes (f), and mitotic spindles (i). (Scale bars: 5 μm.)