MARK4 localizes to the basal body in ciliated cells. (A) RPE1 cells treated with control or MARK4 siRNA were serum starved for 48 h and stained for MARK4, polyglutamylated tubulin, and DNA. (B and C) RPE1 cells were transiently transfected with GFP-hMARK4L and stained for γ-tubulin (B) or acetylated tubulin (C) and DNA. (D) After control or MARK4 depletion, LAP-hMARK4L or LAP-hMARK4L-kd (kinase-dead mutant) expression was induced by the addition of 100 ng/ml doxycycline (dox), the cells were serum starved for 24 h, and percentages of ciliated cells were determined using acetylated tubulin as a cilia marker. Data are means ± SD of four independent experiments. *, P < 0.05. (E) Immunoblot analysis of the indicated NIH 3T3 cell extracts using anti-GFP and anti-MARK4 antibodies. Actin served as a loading control. Asterisks indicate unspecific bands. exo., exogenous; endo., endogenous. In A–C, the right images show merged images. Insets show higher magnification.