ODF2 overexpression rescues cilia loss after MARK4 depletion. (A and B) RPE1 cells were transiently transfected with GFP-hODF2 (A) or GFP (B) and subsequently treated with control or MARK4 siRNA for 24 h before serum withdrawal and incubation for another 24 h. Cells were stained for DNA, γ-tubulin, and acetylated tubulin. The left images show merged images. Regions within the white boxes are shown at a higher magnification on the right. (C) Percentages of ciliated cells based on acetylated tubulin as a cilia marker. UT, untransfected. (D) Efficiency of MARK4 depletion in untransfected and GFP-hODF2–expressing cells were determined by quantitative fluorescence microscopy using MARK4-specific antibodies. Boxes show the top and bottom quartiles (25–75%) with a line at the median, and whiskers extend from the minimum to the maximum of all data. (E and F) HEK293T cells were transiently transfected with the indicated constructs. Immunoprecipitations (IP) were performed using anti-FLAG agarose. (E) Interacting proteins were detected by immunoblotting. (F) Quantification of E. (G) NIH 3T3 cells and NIH 3T3 cells expressing low levels of LAP-MARK4L were serum starved for 24 h, and immunoprecipitations were performed on the lysates using anti-GFP beads and probed for endogenous ODF2 and MARK4. exo., exogenous; endo., endogenous. The asterisk indicates an unspecific band. (H) For in vitro kinase assays, purified 6His-MARK4L, either catalytic active (ca) or kinase dead (kd), was incubated with recombinant ODF2 truncations. GST was used as a negative control. Samples were subjected to SDS-PAGE followed by autoradiography (32P) and Coomassie Brilliant blue staining (CBB). An aliquot of each reaction was analyzed by immunoblotting with anti-MARK4. (I) Relative phosphorylation of ODF2-F1, ODF2-F2, and ODF2-F3 normalized to protein amounts. (J) Schematic representation of ODF2 truncations. A.U., arbitrary unit. Data are means ± SD of three independent experiments. *, P < 0.05; **, P < 0.001.
