FIGURE 4:

Nek8445 regulates microtubule organization. (A) Immunofluorescence staining of acetylated and total α-tubulin. Acetylated tubulin is stained with the anti-acetylated α-tubulin antibody 6-11B-1 (green) and total tubulin is stained with the anti–α-tubulin antibody DM1A (red). Note changes in overall tubulin staining after Nek8445 depletion including reduced ventral disk (vd) staining, the lack of a median body (mb) in rounded cells, and nodes of tubulin staining at the ends of flagella. Scale bar = 5 µm. (B) Quantification of mutant phenotypes observed after Nek8445 depletion (n > 300 cells). (C) Measurement of anterior and caudal axoneme length from control morpholino and Nek8445 knockdown cells (n = 76 measurements from control cells and n = 64 measurements for 8445 knockdown cells). (D) Immunoblotting for total tubulin (DM1A) vs. acetylated tubulin (6-11B-1 antibody) in Nek8445 and control morpholino-treated cells. Lysates were prepared from equal numbers of cells for each replicate. There is no significant difference in the ratio of acetylated to total tubulin although overall tubulin levels are decreased by ∼25% in Nek8445-depleted cells.