Fig. 5: RHBDL4 null MEFs have upregulated β-catenin levels and activity due to decreased proteasomal degradation.

A Total β-catenin and pβ-catenin expression as well as pβ-catenin/β-catenin ratio in R4^−/− and WT MEFs. Expression was quantified and normalized to Ponceau-S. n = 3 biological replicates, mean ± SEM; Two-tailed, unpaired t-test, significant p-values reported. B Total and nuclear β-catenin abundance in R4^−/− and WT MEFs. The nucleus is stained with DAPI (purple) while β-catenin is in green in the merged images. Confocal images taken at different z-planes were summed (step size 0.22 μm). Mean signal intensity per image normalized to cell area was quantified for total β-catenin expression while mean signal intensity normalized per nuclear area was quantified for nuclear β-catenin expression. Representative images of two biological replicates shown, (4 images quantified per genotype, 3 nuclei quantified per image) mean ± SEM; Two-tailed unpaired t-test for total and two-tailed unpaired t-test with Welch’s correction for nuclear, significant p-values reported. C Total β-catenin expression in R4^−/− and WT MEFs after MG132 treatment. Pan-ubiquitin is used as a control for successful proteasomal inhibition. Expression was quantified and normalized to Ponceau-S. n = 3 biological replicates, mean ± SEM; Two-way ANOVA (p = 0.029 for the interaction, p = 0.009 and p < 0.001 for the treatment main effect) with Tukey’s multiple comparison test, significant p values for post hoc analysis reported. Normality assumption tested with Shapiro-Wilk test while homogeneity of variance tested with F-test for t-tests and Brown-Forsythe test for ANOVA analyses.