Figure 5.

MRTF and SRF are present in isolated cilia and interact with the components of the deciliation machinery

(A) Primary cilia were isolated from LLC-PK1 cells as described in STAR Methods. Left panel: An aliquot of the preparation was layered onto a coverslip, fixed, stained with acetylated tubulin, and visualized by immunofluorescence microscopy. Note the compact tubular and vesicular acetylated tubulin-positive structures, indicating large enrichment of PC-derived material. Right panel: Quality control of the cilium preparation. Equal amount of protein (5 μg) from cilium preparations and the non-ciliary fraction (residual whole cell lysates) of serum-deprived cells were analyzed by Western blotting using ciliary (Ac-Tub, Arl13b), nuclear (histone), nuclear/cytosolic (Nup98) and cytosolic (GAPDH) markers. Note the robust enrichment of ciliary markers and the absence of the nuclear marker in the cilium preparation.

(B) The effect of serum stimulation on the level of indicated proteins in PC preparations as determined by Western blotting. LLC-PK1 cells were serum-deprived for 48 hr, and then left untreated or exposed to 10% serum-containing medium for 2 hr. Note that serum induces substantial recruitment of MRTF and increases SRF phosphorylation in the PC.

(C) LLC-PK cells were transfected with siNR or siMRTF for 24 hr, serum-deprived for 48 hr, and then incubated for 3h in the absence or presence of serum. Primary cilia were then isolated from each cell population. The ciliary content of the indicated proteins (Input) is shown in the left panel. These preparations were then subjected to immunoprecipitation with an anti-SRF antibody (IP SRF), and the precipitates were probed for SRF and the indicated proteins, shown in the right panel.

(D–I) Quantification of the indicated proteins in cilium fractions isolated from control (NR) or MRTF-silenced (MRTF) cells, under serum-free (SF) or serum-stimulated (Ser) conditions. The average density obtained from the control fractions (siNR-transfected cells, SF condition) was set to 1 for each protein, and means ± SD are shown. ∗∗∗p < 0.0005, ∗∗∗∗p < 0.0001, determined by ANOVA; & p < 0.05, &&&& p < 0.0001 when fold changes were compared only in the corresponding two groups by ratio t test.

See also Figure S5 for changes in each individual experiment.