G9295

Tissues were lysed in ice cold RIPA buffer (Sigma) together with protease (Complete Mini, Roche) and phosphatase inhibitors (Thermo scientific, Waltham, MA, USA). For further analysis, the following primary antibodies were used; anti-NCALD (1:1,000, 12925-1-AP, Proteintech), anti-beta-actin (1:2,500, A5316, Sigma), anti-GAPDH (1:5,000 G-9295, Sigma), anti-myelin basic protein (anti-MBP; 1:1,000 SMI94, Covance), anti- MAP3K10 (1:500, NBP1-87737, Novus Biologicals), anti-pJNK (1:500, sc-6254, Santa Cruz), anti-JNK (1:1,000, #9252, Cell signaling). Chemiluminescence signal was detected with HRP conjugated-secondary antibodies and Chemiluminescence reagent (Thermo Scientific, Waltham, MA, USA) according to manufacturer’s protocol.

Both wild-type and R350P/R349P mutant desmin proteins were detected by two commercially available desmin antibodies [mouse monoclonal antibody (mAb), D1033, Sigma-Aldrich (St. Louis, MO, USA), 1:1000 in TBS-T for western blotting; rabbit polyclonal antibodies (pAb), #10570, Progen Biotechnik GmbH (Heidelberg, Germany), 1:100 in PBS for immunofluorescence], GAPDH by a mouse mAb [G9295, Sigma-Aldrich (St. Louis, MO, USA), 1:10,000 in TBS-T for western blotting], multiple complex I subunits by a rabbit anti-serum ([8 (link)], antibody #55, purified bovine complex I was used for immunization, 1:10,000 in TBS-T for western blotting), complex II (SDHA) by a mouse mAb (#459200, Invitrogen, 1:1000 in TBS-T for western blotting), complex III [UQCRC2 subunit (Core protein II)] by a mouse mAb (A11143, Molecular Probes, 1:3000 in TBS-T for western blotting), complex IV (CoxVIa subunit) by a rabbit anti-serum ([30 (link)], antibody #90, 1:10,000 in TBS-T for western blotting), complex IV (CoxI subunit) by a mouse mAb (#59600, Invitrogen, 1:100 in PBS for immunofluorescence), complex V (ATP5A subunit) by a mouse mAb (#459240, Invitrogen, 1:1000 in TBS-T for western blotting), and multiple subunits of complexes I–V by a ready-to-use mixture of mouse mAbs (MS604, MitoSciences, 1:5000 in TBS-T for western blotting).

Total protein of the ACC tissues was extracted using RIPA buffer supplemented with a protease inhibitor cocktail. Then, the concentration of protein was determined using the BCA Protein Assay Kit (catalog no. P0010S, Beyotime). Subsequently, 30 μg of protein was loaded per lane. After gel electrophoresis, membrane transfer, and blocking, the membranes were immunoblotted with primary antibodies against 5-HT_1ARs (1:1,000, catalog no. ab85615, Abcam), 5-HT_2ARs (1:100, catalog no. sc-166775, Santa Cruz), 5-HT₇Rs (1:1,000, catalog no. ab128892, Abcam), and GAPDH (1:10,000, catalog no. G9295, Sigma-Aldrich) at 4^°C overnight. After washing, the membranes were incubated with horseradish peroxidase-labeled goat anti-rabbit IgG antibody (1:2,000, catalog no. ARG65351, Arigo) or horseradish peroxidase-labeled goat anti-mouse IgG antibody (1:2,000, catalog no. ARG65350, Arigo) at room temperature for 2 h. Membranes were revealed using enhanced chemiluminescence reagents (ECL, catalog no. WBKLS0010, Millipore) by a chemiluminescence imaging system (GE Healthcare, United States). The protein expression levels were quantified based on the intensities of the bands with the Image J software and normalized against the GAPDH expression level. Additionally, the membranes reacted with 5-HT receptor antibodies, then stripped, and re-probed with GAPDH antibody in western blot analysis.

Gel electrophoresis was run with NuPAGE 4–12% Bis–Tris pre-cast gels and the accompanying buffers (Invitrogen) following the manufacturer’s instructions. Antibodies against BMPR2 (1:2000, Ma5-15827, Thermo Fisher Scientific), pSMAD1/5/9 (1:1000, 13820, Cell Signaling), pSMAD2 (1:1000, gift from Prof. ten Dijke at LUMC Leiden), and GAPDH (1:10000, g9295, Sigma-Aldrich) were used for protein detection.

The following antibodies were used, anti-TFEB rabbit antibody (Cell Signaling Technology, D207D), anti-RPS6KB/S6K rabbit antibody (Cell Signaling Technology, 9202), anti-phospho-RPS6KB/p70 S6 kinase (Thr389) rabbit antibody (Cell Signaling Technology, 9205), anti-EIF4EBP1 rabbit antibody (Cell Signaling Technology, 9452), and anti-phospho-IEF4EBP1 (Thr37/46) rabbit antibody (Cell Signaling Technology 9459), mouse anti-human HTT/huntingtin clone 1HU-4C8 (Sigma-Aldrich, MAB2166), anti-LC3 antibody (MBL International, PM036), rabbit anti-FOXO3/FOXO3a for immunohistochemistry (Cell Signaling Technology 12,829), monoclonal anti-FOXO3 for western blotting (Cell Signaling Technology 99,199), and monoclonal anti-phosphorylated FOXO3 for western blotting (Cell Signaling Technology, 8174). The anti-mouse and anti-rabbit antibodies, as well as anti-GAPDH antibodies, were from Sigma Aldrich (A9044, A0545, and G9295, respectively).

Cells were lysed using lysis buffer (Millipore) with Protease Inhibitor Cocktail (Roche). Protein quantification was performed using a BCA Kit (Beyotime). Protein lysate was subjected to SDS‐PAGE and subsequently electrotransferred onto a polyvinylidene fluoride membrane (Millipore). Blots were developed by indicated antibodies and enhanced chemiluminescence (ECL) (Millipore, WBKLS0500), followed by a ChemiScope Mini chemiluminescence imaging system (Clinx Science). The antibodies used are listed as follows: anti‐GPx7 (Abclone, A3902, 1:1,000), anti‐Nrf2 (Abcam, ab62352, 1:1,000), anti‐PDI (Abcam, ab2792, 1:2,000), anti‐GPx8 (GeneTex, GTX125992, 1:1,000), anti‐Prx4 (Animal Facility, Institute of Genetics and Developmental Biology, CAS, rabbit serum, 1:500), anti‐ERp44 (CST, 3798, 1:2,000), anti‐Ero1α (Millipore, MABT376, 1:1,000), anti‐ERp46 (Animal Facility, Institute of Genetics and Developmental Biology, CAS, rabbit serum, 1:500), anti‐ERp72 (Origene, TA503904, 1:2,000), anti‐GAPDH (Sigma, G9295, 1:50,000), anti‐Flag (Sigma, F1804, 1:4,000), anti‐α‐tubulin (Sigma, T6074, 1:10,000), goat anti‐rabbit IgG (Sigma, A0545, 1:10,000), goat anti‐mouse IgG (Sigma, A4416, 1:10,000).