Western blotting detection reagent

Western blot was used for analysis of OB and OBR proteins in testis, epididymal tissue, ejaculated and epididymal sperm of rams as described by the method of Zhang (Zhang et al., 2017 (link)). Briefly, tissues or cells were lysed with a lysis buffer (HX1862; Huaxingbo, Hong Kong, China). Equal amounts of protein were resolved using 12% SDS-PAGE gel and transferred to PVDF membranes (ISEQ00010; Millipore, Burlington, MA, USA). After they were blocked with 5% nonfat milk or BSA (A3311; Sigma-Aldrich, St. Louis, MI, USA) at 37 °C for 60 min, the membranes were incubated with primary antibodies against OB (1:1,000, ab3583, Abcam, Cambridge, UK), OBR (1:1,000, 20966-1-AP, Proteintech, Rosemont, IL, USA) and GAPDH ( 1:2,500, WL01114; Wanleibio, Hebei, China) at 4 °C, overnight. The membranes were then washed 3–5 times with TBST buffer (HX1893; Huaxingbo, Hong Kong, China) and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5,000, 7074; Cell Signaling Technology, Danvers, MA, USA). Western blotting detection reagents (GE Healthcare, Uppsala, Sweden), and then were visualized using a cooled CCD camera (Nikon, Tokyo, Japan) based chemiluminescence detector (GE Healthcare, Uppsala, Sweden). Protein bands were then analyzed by computer software (Image-Quant 350; GE Healthcare, Uppsala, Sweden).

Tissue samples were homogenized in liquid nitrogen and dissolved in buffer containing 250 mM sucrose, 10 mM triethanolamine, protease inhibitor (cOmplete™; Roche) as previously described (Seidel et al., 2012 (link); Neymeyer et al., 2015 (link)). Samples were sonicated and nuclei removed by centrifugation (1,000 ×g for 10 min). Postnuclear supernatants were separated on 10% polyacrylamide mini-gels. Proteins were transferred to nitrocellulose membranes. Unspecific binding sites were blocked with 5% skim milk in PBS for 1 h at room temperature. Antibodies were diluted in milk as detailed in Supplementary Table S1 and applied overnight at 4°C. The abundance of α-tubulin was determined in parallel and served as loading control. Detection of bound primary antibodies was performed by incubation with horseradish peroxidase-conjugated secondary antibodies (Agilent Technologies, Santa Clara, United States) diluted in milk. Signals were generated by incubation with ECL solution (Western Blotting Detection Reagents, GE Healthcare). Image acquisition and signal quantification were performed using an Intas ECL ChemoCam Imager HR 3.2/6.0 (Intas Science Imaging Instruments, Göttingen, Germany).

The same OD equivalents of previously harvested samples were prepared for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Firstly, the proteins were separated by SDS-PAGE and transferred onto the polyvinyl difluoro (PVDF) membranes (Millipore, Bedford, MA, USA) using a Mini Trans-Blot system (Bio-Rad) [10 (link)], which was then followed by 4 °C overnight incubation in 5% BSA. The next day, the membranes were washed three times 10 min with PBST buffer before incubated with the Strep-tag antibody (1:5000; IBA) at room temperature for 90 min, and then the membranes were washed twice with PBST. Finally, the signal density was visualized using the freshly mixed Western blotting detection reagents (GE Healthcare Life Sciences) and detected by the Molecular Imager ChemiDoc XRS+ (Bio-Rad).

Western blotting was performed using standard protocols. In brief, nuclear proteins were isolated using a nuclear extraction kit (Active Motif, Carlsbad, CA, USA). In addition, cell lysates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to a polyvinylidene fluoride membrane, and blocked in 5% skimmed milk. Membranes were incubated overnight at 4°C with primary antibodies: anti-rabbit P-P65 (1:1,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) or anti-rabbit Lam B1 (1:1,000; Miao Tong Biological Science & Technology Co., Ltd., Shanghai, China). An anti-rabbit secondary antibody (1:2,000; Miao Tong Biological Science & Technology Co., Ltd.) was added to membranes at room temperature (RT) for 1 h. The specific protein bands were visualized using enhanced chemiluminescence plus Western blotting detection reagents (GE Healthcare Life Sciences, Pittsburgh, PA, USA) and an LAS4000 luminescent image analyzer (Kodak, Rochester, NY, USA).

The sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE)-separated proteins were electrophoretically transferred to a polyvinylidene difluoride (PVDF) membrane (Immobilon, Merck, Darmstadt, Germany). The membranes were blocked (Blocking One, Nacalai Tesque, Kyoto, Japan) and then incubated with the following primary antibodies: anti-CMA (AGE-M04, Cosmo Bio, Tokyo, Japan), anti-RAGE (sc-365154, Santa Cruz Biotechnology, Dallas, TX, USA), and anti-β-actin (20536-1-AP, Proteintech, Rosemont, IL, USA). Horseradish peroxidase-conjugated secondary antibodies (SA00001-1 and SA00001-2, Proteintech) were used. Detection was performed by chemiluminescence using enhanced chemiluminescence (ECL) Western Blotting Detection Reagents (GE Healthcare, Little Chalfont, UK Life Science, Chicago, IL, USA) and Western Lightning Ultra (Perkin Elmer, Waltham, MA, USA). The detected CMA and RAGE band intensities were analyzed and quantified using the Image Studio™ analysis software (LI-COR Biosciences, Lincoln, NE, USA).

Quinacrine dihydrochloride (Qa) (69–05–6, Sigma-Aldrich, St. Louise, MO), LY411575 (GSI) (a kindly gift by Dr. Golde, Mayo Clinic, Jacksonville, FL), neuroblastoma cells expressing 6× PrP^C, N2a.cl3, (a kindly gift from Dr. Ghaemmaghami, UCSF, San Francisco, CA)[41] (link), gentamycin, 50 mg/ml, penicillin-streptomycin, 10,000 units/ml and 10,000 µg/ml, respectively, GlutaMAX, Dithiothreitol (DTT) and proteinase K (PK), Hoechst (Invitrogen), Complete protease inhibitor cocktail tablets (Roche, Indianapolis), Western blotting detection reagents (GE Healthcare), anti-MAP2 antibody (AB5622, Millipore), anti-GFAP (20334, Dako), anti-actin (MA515739, Thermo Scientific), anti-PrP antibodies, R2, D18, D13, HumP, and D13 conjugated with horseradish peroxidase (HRP) (from Dr. Prusiner, UCSF, San Francisco, CA), Donkey anti-rabbit IgG conjugated with Alexa488 and Donkey anti-human IgG conjugated with Alexa594 (Jackson Immunoresearch Laboratory) and AAV2 constructs (Virovek, Hayward, CA).