FIGURE 8.

Npas4 is degraded via the ubiquitin-proteasome pathway. MIN6 cells were kept in either basal medium alone (Basal) or with 40 mm KCl (2 h KCl) for 2 h in the presence of 10 μm MG132 or vehicle control. A–D, MIN6 cells were subjected to either stimulation in KCl medium (+ KCl, A and B) or basal medium washout (+Basal, C and D). Blocking the proteasome with MG132 prevented the gradual degradation of Npas4 protein after 3 and 6 h of KCl treatment (A and B, conditions 3 and 4 + MG132). Addition of MG132 also prevented Npas4 degradation following 1 h in basal medium, but NPAS4 levels declined modestly after 3 h of basal washout (C and D, conditions 3 and 4 + MG132). E, MIN6 cells were cultured in basal medium (1), treated for 2 h with 40 mm KCl (2) or KCl, followed by a 1-h washout in basal medium (3), all in the presence of 10 μm MG132. Cells were lysed and immunoprecipitated with an ubiquitin-specific antibody (IP α Ub) and then Western-blotted. Npas4 immunoreactivity was observed in KCl-treated (2′ and 3′) but not unstimulated (1′) IP fractions. n = 3. Error bars represent mean ± S.E., and significance was determined using a one-way ANOVA with Bonferroni's multiple comparison test. *, p ≤ 0.05; ***, p ≤ 0.001.