A1978

Collected cells were washed with 1× PBS buffer, prepared with RIPA buffer supplemented with protease/phosphatase inhibitor cocktail, and centrifuged at 12,000 r/min for 5 min at 4°C to yield the total protein extract in the supernatants. The protein concentration was measured with a BCA assay kit (Beyotime Institute of Biotechnology) according to the manufacturer's protocol. Equal amounts of protein were separated by 8% SDS‐PAGE and subsequently transferred to polyvinylidene difluoride membranes (Millipore). Membranes were blocked with 5% nonfat milk in Tris‐buffered saline with Tween^®20 (TBST) for approximately 1 hr, followed by incubation with primary anti‐GAPDH (1:5,000; G8795, Sigma‐Aldrich), anti‐β‐actin (1:5,000; A1978, Sigma‐Aldrich), and rabbit polyclonal anti‐POU6F2 (1:1,000, TA351549, OriGene Technologies) antibodies overnight at 4°C. After washing with TBST, membranes were incubated with horseradish peroxidase conjugated secondary antibody (Cell Signaling Technology) at room temperature for 1 hr. ImageJ (NIH) was used to quantify the protein band densities.

The PKCα protein expression level was determined in the spinal cord of the rats injected with AS- or MS ODNs (n = 6/group). The tissue from the dorsal spinal cord was dissected out and immediately frozen in liquid nitrogen. The tissue was then homogenized in an ice-cold RIPA-buffer (1:3) that contained 20 mM Tris–HCl, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, 1 µM leupeptin and 1 mM protease inhibitor PMSF (pH 7.5). The homogenate was centrifuged (15 min at 11,000×g, 4 °C) and the supernatant was collected. After measuring the protein concentration, the protein fraction was separated using 10% polyacrylamide gel with 0.1% SDS and then electrophoretically transferred onto a nitrocellulose membrane (90 min at 200 mA). For the Western blot analysis human/mouse/rat affinity purified polyclonal antibody for PKCα (1:1000, incubation for overnight, 4°С, CAF5340-SP, R&D Systems Inc, UK) and monoclonal mouse primary antibody for β-actin (1:1000, 2 h incubation, A1978, Sigma, USA) were used. The proteins were detected with anti-goat or anti-mouse secondary antibodies and visualized with chemiluminescence reagents provided with the ECL kit (Amersham Pharmacia Biotech, Piscataway, NJ) and exposured to film. The PKCα protein level, normalized to the corresponding β-actin value, was calculated for each individual sample as the relative expression (to naïve group).

Proteins were separated by SDS-PAGE in 10% gels and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with PVDF Blocking Reagent for Can Get Signal (Toyobo, Osaka, Japan) for 1 h. The membranes were incubated with primary antibodies against mouse GCAT (1:1,000; sc-86466, Santa Cruz Biotechnology, Dallas, TX, USA), mouse SHMT2 (1:1,000; #12762, Cell Signaling Technology, Danvers, MA, USA), β-ACTIN (1:10,000; A1978, Sigma, St. Louis, MO, USA) or α-TUBULIN (1:50,000; T5168, Sigma, St. Louis, MO, USA) for 1 h at room temperature; Can Get Signal Immunoreaction Enhancer Solution 1 (Toyobo) was used for dilution. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies against goat IgG (1:20,000; HAF109, R&D Systems, Minneapolis, MN, USA), rabbit IgG (1:10,000; G-21234, Thermo Fisher Scientific, Waltham, MA,USA) or mouse IgG (1:10,000; G-21040, Life Technologies, Carlsbad, CA, USA) for 1 h at room temperature; Can Get Signal Immunoreaction Enhancer Solution 2 (Toyobo) was used for dilution. Bands were detected with a bio-imaging analyser, EZ-Capture ST (ATTO, Tokyo, Japan) using ECL Select Western Blotting Detection Reagent (GE Healthcare, Buckinghamshire, UK).

GFP (Roche, 11814460001; 1:2000), PAR 10H (Trevigen, 4335-AMC-050; 1:200), PAR binder (Millipore, MABE1031, 1:1000), CTIF (Sigma-Aldrich, HPA016865-100UL; 1:1000), TERF1 (Abcam, ab10579; 1:1000), GLUT4 (Abcam, ab654; 1:1000), ILF3 (Abcam, ab92355; 1:1000), NPM1 (Abcam, ab10530; 1:1000), CETN3 (Abnova, H00001070-M01; 1:500), PCNT (Atlas antibodies, HPA016820; 1:500), PCM1 (Cambridge bioscience, A301-149A; 1:500), gamma-tubulin (Sigma-Aldrich, T6557-100UL; 1:500), Azi1/Cep131 (Abcam, ab84864; 1:500), Cep290 (Abcam, ab84870; 1:500), BBS4 (Proteintech, 12766-1-AP; 1:500), OFD1 (Proteintech, 22851-1-AP; 1:500), TNKS1/2 (Santa Cruz, sc-8337; 1:1000), alpha-tubulin (Sigma-Aldrich, T9026; 1:10000), beta-actin (Sigma-Aldrich, A1978; 1:10000).

Samples containing equal amounts of protein (20 μg) from lysates of the rat testes or cultured TM4 cells were subjected to SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose filter. The filter was blocked in PBS containing 5% nonfat milk powder at 4°C overnight and then incubated for 1 hour with antibodies against nephrin (1 : 200, sc-32532, Santa Cruz Biotechnology, Santa Cruz, California), podocin (1 : 200, P0372, Sigma-Aldrich, Tokyo, Japan), and β-actin (1 : 1000, A1978, Sigma-Aldrich). The filters were then incubated for 30 minutes with horseradish peroxide-conjugated secondary antibodies (1 : 2000, sc-2004, sc-2020 Santa Cruz Biotechnology), and specific proteins were detected using an enhanced chemiluminescence Western blot analysis system (RPN2106, GE Healthcare, Tokyo, Japan). The relative optical densities of the bands were quantified using Image J Program [29 (link)], and data were expressed as corresponding values of the ratio relative to the result from control group.

Skeletal muscle and fibroblast homogenates were obtained according to previously described methodologies.³⁰ (link) 30–40 μg (S1–S3) and 20 μg (S4) of whole-cell protein extracts were separated by SDS polyacrylamide (12%) electrophoresis and then wet transferred to polyvinyl difluoride (PVDF) membranes. For S4, a 4%–12% gradient gel was used. Immunological detection of proteins was carried out with the following primary antibodies: C1QBP (ab24733, Abcam), β-actin (A1978, Sigma), α-tubulin (ab7291, Abcam), and OXPHOS complex-specific antibodies (NDUFS3 [ab14711, Abcam], NDUFB8 [ab110242, Abcam], NDUFA9 [MS111, Molecular Probes], SDHA [459200, MitoSciences], SDHB [ab14714, Abcam], UQCRC2 [ab14745, Abcam], COXI [ab14705, Abcam], COXII [ab110258, Abcam], COXIV [ab14744, Abcam], and ATP5A [ab14748, Abcam]). Species-appropriate horseradish-peroxidase-conjugated secondary antibodies (DAKO, P0399, and P0260) were used.