Fig. 4
Loss of UBR5 impairs HTT protein levels in iPSCs. a qPCR analysis of UBR5 and HTT mRNA levels in control iPSCs #1 (n = 4 independent experiments), HD Q71-iPSCs #2 (n = 3) and HD Q180-iPSCs (n = 3). Graphs (relative expression to non-targeting (NT) shRNA) represent the mean ± s.e.m. b, c Western blot analysis of the indicated control iPSC lines with antibodies to HTT and polyQ-expanded proteins. Proteasome inhibitor treatment: 5 µM MG-132 for 12 h. The graphs represent the HTT relative percentage values to DMSO-treated NT shRNA iPSCs corrected for β-actin loading control (mean ± s.e.m. of three independent experiments). d Western blot analysis of HD Q71-iPSC line #2 upon UBR5 knockdown. The graphs represent the relative percentage values to DMSO-treated NT shRNA iPSCs (corrected for β-actin) of nHTT and mHTT detected with antibodies to total HTT and polyQ-expanded proteins (mean ± s.e.m. of four independent experiments). Proteasome inhibitor treatment: 5 µM MG-132 for 12 h. e Western blot analysis of HD Q180-iPSCs upon UBR5 knockdown. Proteasome inhibitor treatment: 5 µM MG-132 for 12 h. The graphs represent the relative percentage values to DMSO-treated NT shRNA iPSCs (corrected for β-actin) of nHTT and mHTT detected with antibodies to total HTT and polyQ-expanded proteins, respectively (mean ± s.e.m. of three independent experiments). f Proteasome activities in HD Q71-iPSCs #1 upon UBR5 knockdown (relative slope to NT shRNA iPSCs). Graphs represent the mean ± s.e.m. of three independent experiments. g Co-immunoprecipitation with UBR5, UBE3A and FLAG antibodies in HD Q71-iPSC line #1 followed by western blot with HTT, polyQ-expanded HTT, UBR5 and UBE3A antibodies. The images are representative of three independent experiments. All the statistical comparisons were made by Student’s t-test for unpaired samples. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001
