Figure S2. Spastin is K48-polyubiquitinated.

(A) Data quantification of spastin/loading control levels by Western blot (WB) on total cell extract (TCE), relative to data shown in Fig 2B. Quantification was performed as in Fig 2A. Bars are mean ± SD of three independent experiments; Statistical differences, ANOVA test. (B) As in Fig 2D, HeLa cells were transfected with vector expressing Ub-HA and treated 24 h posttransfection with 20 μM MG132 for 8 h. TCE was immunoprecipitated with anti-spastin mouse monocolonal Ab, sp3G11/1. Mouse IgG were used as negative IP control. TCE and IPs were analysed by WB with anti-HA and anti-spastin Abs. The arrow indicates the position of the unmodified spastin and the asterisk indicates a non-specific band. (C, D) HeLa HIPK2-Cas9 and Ctr-Cas9 cells were transfected with vectors expressing HA-Ub or its derivative K48-Ub-HA (i.e., Ub with only K48, the other lysines are mutated in arginines) and treated 24 h post transfection with 20 μM MG132 or DMSO for 8 h. TCE were analysed by WB and IP as in Fig 2D. In (C), representative WB of three independent experiment is shown. The arrow indicates the position of the unmodified spastin and the asterisk indicates a non-specific band. In (D), relative spastin-Ub-HA levels were quantified and reported as in Fig 2C. Statistical differences, unpaired t test. (E) As in Fig 2F, HeLa cells were transfected with vectors expressing flag-myc tagged spastin in combination with Ub-HA and 24 h post transfection cells were treated with 20 μM MG132 for 8 h. TCE was immunoprecipitated with anti-Flag mouse monoclonal Ab. Mouse IgG were used as negative IP control. TCE and IPs were analysed by WB with anti-HA and anti-Flag Abs. The arrow indicates the position of the unmodified spastin.