(A) Representative images of NSC-34 cells transfected with UbG67V-GFP alone, or co-transfected with UBQLN4-WT or UBQLN4D90A. DAPI staining is shown in blue. Scale bar: 100 μm. (B) Quantification of GFP levels in (A) revealed reduced proteasomal turnover following UBQLN4D90A expression. GFP signal, normalized to DAPI, was greater in UBQLN4D90A transfected cells than in UbG67V-GFP-only transfected cells (p=0.0008), or UBQLN4-WT transfected cells (p=0.0088). The difference in GFP level between UbG67V-GFP -only and UBQLN4-WT transfected cells was not significant (p=0.1114). Results are from three independent experiments and are mean ± SEM. ***p<0.001, **p<0.01, one-way ANOVA with Bonferroni post-hoc test. (C) Cycloheximide protein stability assay. GFP protein stability is compared between UbG67V-GFP-only, UbG67V-GFP + UBQLN4-WT, and UbG67V-GFP + UBQLN4D90A transfected NSC-34 cells treated with cycloheximide for 0, 2, 4, and 6 hr. Quantification of GFP level revealed impeded protein turnover in UBQLN4D90A transfected cells. GFP level of UbG67V-GFP + UBQLN4D90A transfected cells was significantly greater than that of UbG67V-GFP + UBQLN4-WT transfected cells at 2, 4, and 6 hr with cycloheximide treatment (p=0.024 (2 hr), p=0.0038 (4 hr), and p=0.036 (6 hr)). GFP level of UbG67V-GFP + UBQLN4D90A transfected cells was significantly greater than that of UbG67V-GFP-only transfected cells (p=0.0068 (2 hr), p=0.0093 (4 hours), and p=0.032 (6 hr)). Results are from three independent experiments and are mean ± SEM. **p<0.01, *p<0.05, one-way ANOVA with Bonferroni post-hoc test. (D) Representative Western blot of the cycloheximide protein stability assay. Actin serves as a loading control. (E) Western blot of beta-catenin levels from UBQLN4-WT, UBQLN4D90A and non-transfected NSC-34 cells. GAPDH Western blot indicates equal protein loading. (F) Quantification of beta-catenin signal in (D) indicated greater beta-catenin levels in UBQLN4D90A transfected and non-transfected cells as compared to UBQLN4-WT transfected cells (p=0.0174 and p=0.0326, respectively). Results are from three independent experiments and are mean ± SEM. *p<0.05, one-way ANOVA with Bonferroni post-hoc test. (G) Representative images of primary mouse neurons transfected with pCAG-GFP and UBQLN4-WT or UBQLN4D90A, stained for beta-catenin. Scale bar: 20 μm. (G) Quantification of beta-catenin localization in (F) revealed increased nuclear localization of beta-catenin in UBQLN4D90A transfected cells as compared to UBQLN4-WT transfected cells (n = 22 or more cells per group, p=0.0038). Data are from three independent experiments and are mean ± SEM. **p<0.01, two-tailed Student’s t-test.