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Figure 1


Fig. 1. IFT88/polaris localizes to basal bodies and axonemes in quiescent ciliated cells and to centrosomes in proliferating cells. (A) RPE-1 cell immunostained with an antibody against acetylated-tubulin (6-11B-1, green) to label the axoneme of the cilium, and with anti-IFT88/polaris (740, red) to stain IFT88/polaris which was localized to the axoneme and the basal body. Insets, higher magnifications of cilium. Ciliary assembly was induced by culturing RPE-1 cells in medium supplemented with 0.25% serum for 48 hours. Bar, 10 µm. (B) Confocal immunofluorescence microscopy analysis, using anti-IFT88/polaris 740 (green) and anti-{gamma}-tubulin (GTU88, red), were performed on exponentially growing RPE-1 cells. Bars, 5 µm. Nuclei in A and B are stained with Hoechst 33342 dye. (C) Left panel, western blot analysis of Triton-X-100-soluble (S) and -insoluble (I) protein fractions from 2A1.6 cells, and of a highly enriched centrosomal fraction (CTR). Right panel, biochemical extraction of centrosome-associated IFT88/polaris. Supernatant (S) and pellet (P) of centrosomal fractions obtained under different extraction conditions (see Materials and Methods) were immunoblotted with anti-IFT88/polaris (02078), anti-{gamma}-tubulin (GTU88) and anti-Akt antibodies. Detection of Akt, a well-characterized cytoplasmic protein, was used as a negative control to verify the purity of the centrosomal fraction.





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