T0198

Isolated proteins from 1 × 10⁶ spermatozoa samples were obtained and immunoblotted as described60 (link). The antibodies used were anti-protein kinase A (9624, Cell Signaling Technology, Beverly, USA, 1:2,000), anti-phosphotyrosine (4G10, Millipore, CA, USA, 1:10,000) and anti-β-tubulin (T0198, Sigma-Aldrich^®, Madrid, Spain, 1:5,000). Blots were visualized by chemiluminescence (Amersham Imager 600, GE Healthcare) using a Pierce^® ECL 2 Western Blotting Substrate (80196, Lumigen Inc, Southfield, MI, USA). The relative amount of signal in each membrane was quantified using the ImageQuant TL v8.1 software (GE Healthcare, Life Sciences, Buckinghamshire, UK).

For Western blot analysis, we used the following: anti-BDNF antibody (1:1000, AB1779SP, Millipore, Burlington, MA, USA); anti-Sig-1R antibody (1:1000, ab53852, Abcam, Cambridge, UK); anti-β-Tubulin antibody (1:1000, T0198, Sigma-Aldrich); goat anti-mouse IgG (H + L) and human ads-HRP (1:500, 1031-05, SouthernBiotech, Birmingham, AL, USA); goat anti-rabbit IgG (H + L); and mouse/human ads-HRP (1:500, 4050-05, SouthernBiotech). For immunohistochemistry, we used the following: living colors® Full-Length GFP polyclonal antibody (anti-GFP) (1:300, 632592, Clontech, Mountain View, CA, USA); donkey anti-Rabbit IgG (H + L) highly cross-adsorbed secondary antibody; and Alexa Fluor 488 (1:500, A21206, Invitrogen).

Reagents and antibodies were obtained from the following sources: anti-αSyn antibody (1:1000; 4B12, GeneTex, Irvine, CA, USA); anti-FABP5 antibody (1:1000; AF3077, R&D Systems, Minneapolis, MN, USA); anti-β-tubulin antibody (1:3000; T0198, Sigma-Aldrich, St Louis, MO, USA); anti-VDAC antibody (1:1000; 4866S, Cell Signaling Technology, Danvers, MA, USA); anti-rabbit or mouse IgG antibody conjugated with horseradish peroxidase (1:5000; Southern Biotech, Birmingham, AL, USA); anti-goat IgG antibody conjugated with peroxidase (1:5000; Rockland Immunochemical, Limerick, PA, USA); Alexa 594-labeled anti-mouse IgG, Alexa 488-labeled anti-Goat IgG and Alexa 594-labeled anti-rabbit IgG antibody (1:500; Invitrogen, Waltham, MA, USA); Biotin-SP anti-Goat IgG antibody (1:500; Jackson ImmunoResearch, West Grove, PA, USA); and AMCA streptavidin (1:500; Jackson ImmunoResearch). These materials were used according to the manufacturers’ datasheets. Rotenone was purchased from MP Biomedicals (150154; Santa Ana, CA, USA). All other reagents were obtained from FUJIFILM Wako Pure Chemicals (Osaka, Japan) unless otherwise noted. The FABP5 ligand (ligand 7) was described in a previous report [24 (link)].

Frozen mouse liver tissue was homogenized in RIPA buffer containing 1× EDTA-free protease inhibitor cocktail (Roche) by using Omni Tissue Homogenizer (Omni International). The concentration of total protein was determined by Bio-Rad Protein Assay and then equalized to 15 μg/μl. 25 μg of total protein was used for western blot assay which was performed as previously described^{24 (link)}. Antibodies used in the western blots are anti-BMAL1 (Cell signaling, #14020, 1:1000), anti-HNF4A (Abcam, ab181604, 1:1000), and anti-TUBULIN (Sigma-Aldrich, T0198, 1:1000).

Purified axonemes in HMDEK buffer (30 mM Hepes–KOH (pH7.4), 5 mM MgSO₄, 1 mM dithiothreitol, 1 mM EGTA, 50 mM potassium acetate, protease inhibitor cocktail (Nacalai Tesque)) were incubated with anti-beta-tubulin antibody (1:10,000 final; T0198, SIGMA) for 15 min on ice, followed by incubation with goat anti-mouse IgG (H+L) 15 nm gold (1:20 final; BB International) and 15 nm colloidal gold conjugated with BSA. The mixtures were loaded onto home-made holey carbon grids and plunged into liquid ethane at −180 °C using an automated plunge-freezing device (EM GP; Leica).