Figure 1. Sp4 protein abundance is regulated by membrane potential and the ubiquitin-proteasome system.
(A) Sp4 polyubiquitination was determined in lysates from CGNs cultured in the presence or absence of the proteasome inhibitor MG132 and either 25mM KCl (normal culture condition) or switched to 5mM for 30 min. Lysates were immunoprecipitated with anti-Sp4 and Western blotted with anti-Ub or anti-Sp4 as indicated. (B) CGNs treated with the 26S proteasome inhibitor MG132 (10μM) or the calpain inhibitor ALLN (20μM) and Sp4 levels determined by Western blot. Sp1 was used as a negative control. (C) Western analysis of Sp4 from CGNs treated for 60 min with fresh culture medium supplemented with the indicated concentrations of KCl. The band specific to Sp4 (marked by arrowhead) migrates just above the 100 kD marker. (D) Graph shows mean (n = 6) Sp4/GAPDH ratio (±SEM) for cells treated as in (C). One-way ANOVA revealed a significant difference between KCl groups [F2,15 = 17.69, p < 0.0001]. Tukey's HSD post hoc test, *p < 0.05; ****p < 0.0001. (E) Sp4 mRNA expression in CGNs treated for 60 min with three different concentrations of [K+]EXT. Changing [K+]EXT did not affect Sp4 mRNA expression in CGNs. Bars represent mean fold-change measured by RT-qPCR (error bars indicate range from three biological replicates).