Figure 3. Kinase activity of HIPK2 regulates spastin protein levels.

(A) Representative Western blot (WB) of HeLa (right panels) and NSC34 cells (left panels) transfected with vectors expressing HA-tagged HIPK2-WT, its derivative -K228A mutant or HA-alone (empty). Statistical differences, Anova test. (B) Representative WB of HeLa cells transfected with vectors expressing flag-myc spastin-WT or indicated spastin mutants in combination with peGFP vector at 10:1 molar ratio and lysed at indicated time post transfection. GFP expression was used as internal control for transfection efficiency. The intensity of spastin-Flag bands was normalized by the intensity of GFP and reported relative to spastin-WT for each time point. Statistical differences, ANOVA test. (C) HeLa cells were transfected with vectors expressing flag-myc–tagged spastin-S268A or spastin-WT, treated with 25 μg/ml cycloeximide 36 h posttransfection and analysed by WB at indicated times after treatment. Note that to minimize differences in spastin levels at the time 0, cells were transfected with different doses of the expressing vectors, that is, 1 μg of spastin-S268A–expressing vector and 0.5 μg of spastin-WT–expressing vectors. Representative WB is shown. The levels of spastin-Flag bands relative to those of GAPDH were measured at each time point and reported as mean ± SEM of four different independent experiments in the right panel. Statistical differences were calculated and reported for each time point, unpaired t test.

Source data are available for this figure.