Fig. 4.

Spastin mutants increase the level of acetylated tubulin in HeLa cells. (A) HeLa cells were transfected with GFP-tagged spastin mutants, fixed after 16 h and stained for acetylated tubulin. Scale bars: 20 µm. (B) Quantification of the average intensity of acetylated or tyrosinated tubulin in GFP-positive cells normalized to nontransfected cells (100%). The number of cells analyzed ranged between 40 and 200. (C) Representative western blot of HeLa cells transfected with the same GFP-tagged constructs as in panel B (note that the blot was stripped and re-probed for Fig. 5E). Increase of acetylated tubulin by treatment with the deacetylase inhibitors TSA or tubacin are shown for comparison. The arrowhead indicates M1CY-GFP, dash indicates M85CY-GFP and circle indicates M1Δ-GFP. (D) Quantification of the ratio between acetylated tubulin and GAPDH from three independent experiments. Data were normalized to untransfected cells (control). Data are shown as mean±s.e.m. Significance was determined by one-way ANOVA, Dunnett's post test. *P<0.05; **P<0.01; ****P<0.001; ns, not significant.