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Fig. 3. Centrosomal assembly of IFT88/polaris is not dependent on polymerized microtubules or the dynein-dynactin molecular motor. (A) Hela cells were either mock-treated (no drug) or treated with nocodazole for 1 hour and allowed to recover for 0 or 5 minutes. Cells were stained with -tubulin antibody (methanol fixation, Tub2.1, green) to follow depolymerization (0 minutes) or polymerization (5 minutes after recovery) of microtubules, or co-stained with anti-IFT88/polaris 740 (PFA 4% fixation, green) and anti--tubulin (PFA 4% fixation, red) antibodies. IFT88/polaris and -tubulin localizations were analyzed on cells treated or not with the nocodazole (n=200). Bars, 10 µm. (B) HeLa cells were transiently transfected with DsRed-p150^217-548, which inhibits dynein-dynactin motor function, and co-stained with antibodies against PCM1, –tubulin or IFT88/polaris (green). The left panel shows untransfected cells with expected PCM1 localisation (greens staining, arrow) and transfected DsRed-p150^217-548 cells with PCM1 mislocalization (scattered staining). Middle and right panels show that, after expression of plasmid DsRedp150^217-548, centrosomal localization in transfected cells is unchanged for -tubulin and IFT88/polaris (arrows). Bars, 10 µm.

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