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Fig. 9 Proteasomal inhibition reverses NEK1 downregulation in C21ORF2-V58L motor neurons. A Immunocytochemistry for NEK1 in MNs from two control (C1, C2) lines and two ALS patient lines carrying C21ORF2 mutations (P1, P2) treated with the proteasome inhibitor MG132 (100 nM, 3 h) or non-treated at days in vitro 7 (DIV7). B Quantification of immunostaining of NEK1 in control and mutant neurons without or with treatment with MG132 as in A. C-D Western blot analysis of lysates of control and mutant MNs without or with MG132 treatment. Western blot analysis using anti-ubiquitin, anti-NEK1 and anti-β-actin antibodies. Beta-actin was used as loading control. C Representative Western blot. D Quantification of Western blot experiments as in C. Results of 3 (A, B) and 5 (C, D) independent experiments are plotted as means with SEM *p < 0.05; **p < 0.01; ****p < 0.0001. (B) Kruskal–Wallis test with Dunn’s multiple comparison post-hoc test. D One-Way ANOVA, Tukey multiple comparison post-hoc test. Scale bar: A, 10 µm