FIGURE 6.

ROCK-mediated TPPP1 phosphorylation inhibits its delay in the G₁/S-phase transition. A, U2OS cells stably expressing wild-type TPPP1, TPPP1^3Ala/4Ala, TPPP1^3Glu//4Ala, TPPP1^3Ala/4Glu, TPPP1^3Glu/4Glu, or vector were incubated with 1 μg/ml of 5-bromo-2-deoxyuridine (BrdU) for 3 h followed by fixation in 70% ethanol. Fixed cells stained with mouse anti-BrdU-FITC or mouse anti-IgGκ FITC (isotype control) antibodies and PI were analyzed by flow cytometry. Representative dot plots of flow cytometry analyzed cells. Insets represent the BrdU-labeled cell population showing the number of cells that have transitioned through S-phase during the incubation period. B, graphical representation of the data in A (*, p = 0.025; **, p = 0.0005). Data are expressed as mean ± S.E. of three independent experiments. C, TPPP1-mediated MT polymerization does not affect its inhibition of HDAC6 activity or regulation of MT acetylation. U2OS cell lysates from cell lines described in A were analyzed by immunoblotting. Total lysates were probed with anti-FLAG, anti-acetyl-α-tubulin, and anti-GAPDH (loading control) antibodies. D, immunofluorescence microscopy of cells described in A that were stained for acetylated tubulin (green) and nuclei (blue). Scale bar is 50 μm.