Figure 4(A) Increased expression of HOIP decreases nuclear phospho-c-Jun in primary neurons. Primary mouse hippocampal neurons expressing Htt-Q25 or Htt-Q97 +/- HOIP were analyzed by immunocytochemistry using an antibody against phospho-c-Jun. Data are displayed as mean ± SD and were analyzed by 2 independent Mann-Whitney U-tests, n = 3. *p ≤ 0.05, ***p ≤ 0.001. (B) Htt-Q97-induced toxicity is increased in cells silenced for HOIP. SH-SY5Y cells transiently transfected with Htt-Q25 or Htt-Q97 and either control siRNA (CO) or HOIP siRNA were analyzed by immunocytochemistry using an antibody against phospho-c-Jun. For rescue experiments, HOIP was co-expressed 24 h after silencing. Quantification of Htt-expressing cells with nuclear phospho-c-Jun is based on Student's t test from 3 independent experiments. Data are displayed as mean ± SD unless otherwise stated. *p ≤ 0.05, ***p ≤ 0.001. OTULIN silencing decreases the toxicity of Htt-Q97. SH-SY5Y cells transiently transfected with either control siRNA (CO) or OTULIN siRNA and Htt-Q25 or Htt-Q97 were analyzed by immunocytochemistry using antibodies against phospho-c-Jun (C) Data are displayed as mean ± SD and were analyzed by one-way ANOVA followed by Tukey's Multiple Comparison Test, n = 9-11. *p ≤ 0.05, ***p ≤ 0.001. OTULIN silencing decreases the toxicity of Htt-Q97. SH-SY5Y cells transiently transfected with either control siRNA (CO) or OTULIN siRNA and Htt-Q25 or Htt-Q97 were analyzed by immunocytochemistry using antibodies against cleaved caspase-3 (D). Data are displayed as mean ± SD and were analyzed by one-way ANOVA followed by Tukey's Multiple Comparison Test, n = 9-11. *p ≤ 0.05, ***p ≤ 0.001. (E) The protective effect of HOIP is independent of NF-κB signaling. SH-SY5Y cells transiently transfected with Htt-Q97-GFP and either control vector (CO) or HOIP ± the NF-κB super-repressor IκB-2S/A were analyzed by immunocytochemistry using an antibody against phospho-c-Jun. Data are displayed as mean ± SD and were analyzed by one-way ANOVA followed by Tukey's Multiple Comparison Test, n = 9. *p ≤ 0.05, ***p ≤ 0.001. (F) SDS-soluble Htt-polyQ species are modified by linear ubiquitin chains. HEK293T cells were co-transfected with Htt-Q60-GFP (as a control for specific immunoprecipitation of HA-tagged Htt-Q60), Htt-Q60-HA or Htt-Q60-3R (3 lysine residues replaced by arginines) and the plasmids indicated. Cells were lysed under denaturing conditions (1% SDS), followed by immunoprecipitation of Htt via the HA tag (1% Triton X-100, 0.1% SDS). Immunopurified proteins were detected by Western blotting using the M1 ubiquitin-specific antibody 1E3 and anti-HA. (G) Silencing of OTULIN increases linear ubiquitination of Htt-Q97. A linear ubiquitination assay was performed as described in (F) with HEK293T cells transfected with Htt-Q97-HA and control siRNA or OTULIN siRNA. (H) SDS-insoluble Htt-polyQ species are modified by linear ubiquitin chains. HEK293T cells expressing Htt-Q97-HA or Htt-Q97-GFP were lysed under denaturing conditions in 1.5% SDS. After centrifugation, the pellets containing the SDS-insoluble aggregates (SDS-insoluble fraction) were dissolved in formic acid. Formic acid-dissolved aggregates were analyzed by immunoblotting using the M1 ubiquitin-specific 1F11/3F5/Y102L antibody. (I) Endogenous NEMO co-localizes with Htt-Q97 aggregates. NEMO (red) and Htt-Q97-GFP (green) were analyzed by immunocytochemistry in SH-SY5Y cells. DAPI (blue). Scale bar, 10 µm. (J) Endogenous Optineurin co-localizes with Htt-Q97 aggregates. Optineurin (red) and Htt-Q97-GFP (green) were analyzed by immunocytochemistry in SH-SY5Y cells. DAPI (blue). Scale bar, 10 µm.