Polyvinylidene difluoride pvdf blotting membranes

Testicular tissues at different development stages were homogenized and lysed using a radio immunoprecipitation assay (RIPA) protein extraction kit (Solarbio, Beijing, China), according to the operating instructions. Protein concentrations within the testis samples were determined using a commercial bicinchoninic acid (BCA) Protein Assay kit (Beyotime, Shanghai, China). Twenty micrograms of the denatured protein samples were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene difluoride (PVDF) blotting membranes (Beyotime, Shanghai, China). After blocking in phosphate buffered saline tween-20 (PBST) containing 5% non-fat milk, the membranes were incubated overnight at 4 °C with either rabbit anti-BOLL polyclonal antibody (1:500, Bioss, Beijing, China) or anti-beta-actin polyclonal antibody (1:1500, Bioss, Beijing, China). After washing, the membranes were incubated with goat anti-rabbit IgG/HRP antibody (1:5000, Bioss, Beijing, China). Enhanced chemiluminescence signals were visualized in an X-ray room. This experiment was biologically repeated three times. Band intensities were quantified using AlphaEaseFC software (Protein Simple, Santa Clara, CA, USA). The BOLL protein expression results, from an average across tissues, were presented as bar charts.

Testicular tissues at different development stages were homogenized and lysed using a radioimmunoprecipitation assay (RIPA) protein extraction kit (Solarbio, Beijing, China) and phenylmethanesulfonyl fluoride (PMSF) (Solarbio, Beijing, China), according to operating instructions. Protein concentrations were quantified using a commercial bicinchoninic acid (BCA) protein assay (Beyotime, Shanghai, China). Protein samples were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then were transferred onto polyvinylidene difluoride (PVDF) blotting membranes (Beyotime, Shanghai, China). The membranes were blocked with phosphate buffered saline tween-20 (PBST) containing 5% non-fat milk for 2 h at room temperature, and then incubated with either rabbit anti-GLOD4 polyclonal antibody (1:500; Bioss, Beijing, China) or anti-beta-actin polyclonal antibody (1:1000; Bioss, Beijing, China) at 4 °C overnight. After being washed with PBST, the membranes were incubated with goat anti-rabbit IgG/HRP antibody (1:5000; Bioss, Beijing, China) for 2 h at 37 °C. After being washed with PBST, the protein signals were visualized using NcmECL Ultra reagents (New Cell & Molecular Biotech Co. LTD, Suzhou, China) in an X-ray room. This experiment was biologically repeated eight times. Each biological replicate consisted of two technical replicates.

After transfection of SCs, they were homogenized and lysed using a radioimmunoprecipitation assay (RIPA) protein extraction kit (Solarbio, Beijing, China) according to the manufacturer’s instructions. Protein concentrations were then quantified using a commercial bicinchoninic acid (BCA) protein assay (Solarbio, Beijing, China). Next, the extracted proteins were denatured with 4x protein loading buffer (DTT, Solarbio, Beijing, China), resolved using 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred onto polyvinylidene difluoride (PVDF) blotting membranes (Beyotime, Shanghai, China). Membranes were first blocked with phosphate buffered saline tween-20 (PBST) containing 5% non-fat milk for 2 h at room temperature, followed by incubation with either rabbit anti-TPI1 polyclonal antibody (1:600; Bioss, Beijing, China) or anti-beta-actin polyclonal antibody (1:600; Bioss, Beijing, China) at 4°C overnight. On the next day, membranes were washed with PBST, and then incubated with goat anti-rabbit IgG/HRP antibody (1:5000; Bioss, Beijing, China) for 2 h at 37°C. Finally, they were washed with PBST and the protein signals were visualized using NcmECL Ultra reagents (New Cell & Molecular Biotech Co.LTD, Suzhou, China) in an X-ray room.