Figure 4.

Analysis of spastin-S268 phosphorylation at the midbody. (A) Indicated HeLa cells were fixed and stained with DAPI, anti-β-tubulin, and p-S268 Abs, and analyzed to quantify the IF signal of p-S268 spastin at the midbody. Representative immunostainings of midbody magnification are shown. Bar, 1 μM. Quantification in arbitrary units (a.u.) is reported in the dot plot showing the single measures, as well as, the mean value ± SEM from two different experiments ****p < 0.0001, Mann–Whitney test. (B) Indicated HeLa cells were stained and analyzed as in A. Representative immunostainings of midbody magnification are shown. Bar, 1 µM. Quantification is reported in the dot plot showing the single measures, as well as, the mean value ± SEM from three different experiments ****p < 0.0001, Mann–Whitney test. (C) Dot plot showing the quantification of the p-S268 spastin IF signal in indicated HeLa cells 24 h upon transfection with low doses of vectors expressing GFP-tagged murine HIPK2-WT or its kinase defective derivative. siHIPK2 cells were transfected with indicated vectors 48 h post siRNA transfection performed as in Figure 1A and analyzed 48 h post vector transfection by IF. Note that, in siHIPK2 cells, the expression of murine HIPK2 protein, showing >97% identity with human HIPK2 and fully recapitulating its functions, is not inhibited by RNAi because human-specific siRNAs were used. ***p < 0.001(p = 0.0005) t-test.