Figure 3
KLC1 and KIF5B are found at the base of cilia. Confocal microscopy analysis of hTERT-RPE cells. (A) Cells were transfected to overexpress Myc-tagged KLC1. In addition to cytoplasmic aggregates, a pool of Myc-KLC1 (anti-Myc, green) is found at the base of cilia (anti acetylated α-tubulin, red). (B) In addition to a diffuse cytoplasmic signal, the anti-KLC1 antibody shows an accumulation of endogenous protein at the base of cilia. (C) Similarly, endogenous KIF5B is found in the cytoplasm and also concentrated at the base of cilia. Yellow boxes mark the area that is magnified and showed in panels on the right. DAPI was used to stain nuclei. Scale bars correspond to 10 μm. (D) Scanning electron micrographs showing cilia in hTERT-RPE cells transfected with control (S.CTRL), KLC1 (S.KLC1) or KIF5B (S.KIF5B) stealth RNA oligos. Scale bars correspond to 1 μm. (E,F) The level of tubulin acetylation in control and KLC1 KD cilia was quantified measuring the fluorescence intensity of the signal obtained using the acetylated α-tubulin antibody (green). The anti-ARL13 (red) signal was used to mark the entire length of the cilium. Scale bars correspond to 2 μm. Each cilium was divided in 10 segments from base to tip (see methods) and the mean intensity was computed. For each experiment (KLC1 KD and control) a ten-point intensity profile was computed by averaging the measure of all cilia in each one of the regions of interest. These profiles are shown, normalized by the measure of its first region of interest. Vertical bars plot the 95% confidence interval about the mean.
