Scheme of WT HOIP and HOIP mutants.
Quantification of the co‐localization of WT HOIP or HOIP mutants and Htt‐Q97 aggregates. Data are displayed as mean ± SD and were analyzed by Mann–Whitney U‐test, n = 3.
Htt‐Q97‐GFP (green) was co‐expressed with HA‐tagged WT or HOIP mutants (red) in SH‐SY5Y cells and analyzed by immunocytochemistry using an antibody against HA. DAPI (blue). Scale bar, 10 μm.
Recruitment of HOIP to Htt‐Q97 is decreased in p97/VCP‐deficient cells. p97/VCP‐deficient SH‐SY5Y cells co‐expressing Htt‐Q97 and HA‐HOIP were reconstituted with either WT VCP or VCP∆PIM and analyzed by immunocytochemistry. Data are displayed as mean ± SD and were analyzed by one‐way ANOVA followed by Tukey's multiple comparison test, n = 9.
Recruitment of p97/VCP to Htt‐Q97 is decreased in HOIP KO cells. WT HAP1 cells (control) or HOIP KO HAP1 cells reconstituted with either WT HOIP or HOIP∆PUB were analyzed by immunocytochemistry. Data are displayed as mean ± SD and were analyzed by one‐way ANOVA followed by Bonferroni's multiple comparison test, n = 5.
The interaction of soluble Htt‐Q60 with p97/VCP is dependent on HOIP. WT HAP1 cells or HOIP KO HAP1 cells were transfected with Htt‐Q60‐HA. 48 h after transfection, cells were lysed with 1% Triton X‐100, followed by an immunoprecipitation of Htt‐Q60 via the HA tag. Immunopurified proteins were detected by Western blotting using an anti‐VCP and anti‐HOIP antibody.
0.001.
