Ecl western blotting substrate

Testicular tissues and cells’ proteins were isolated using RIPA lysis buffer (FD008, Fudebio, Hangzhou, China) with PhosSTOP™ (4906845001, Roche, Basel, Switzerland) and 0.1 mM PMSF (FD0100, Fudebio, Hangzhou, China). The BCA Protein Assay Kit (23225, Thermo Fisher Scientific, Waltham, MA, USA) was used to quantify the protein content. Then, 30 ug protein was separated by SDS-PAGE and transferred to the PVDF membrane (10600021, Cytiva, Marlborough, MA, USA). The membrane was blocked with 5% skim milk for 1 h, then incubated with primary antibody overnight and secondary antibody for 1 h. The primary antibodies are listed in Table 3. The membrane was visualized with ECL Western Blotting Substrate (K12045D50, Advansta, San Jose, CA, USA) and the bands were quantified by Image J software (1.48v, National Institutes of Health, Bethesda, MD, USA).

Cells were lysed using an ice-cold lysis buffer with the protease inhibitor PMSF (Beyotime, Nantong, China), and the concentrations of proteins in the supernatant was determined by the bicinchoninic acid (BCA) method. An amount of 10% (w/t) sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate protein (30 μg) samples, which were then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA). An amount of 5% skim milk in Tris-buffered saline (TBST) (containing 0.1% Tween-20) was used to block the membranes for two hours at room temperature. After three 10 min TBST washes, the resulting membranes were incubated overnight at 4 °C with the following primary antibodies: YY1 (ab109237, 1:5000, Abcam, Waltham, MA, USA), p53 (ab32389, 1:10000, Abcam, Cambridge, MA, USA), HDM2 (ab16895,1:500, Abcam, USA), Bcl-2 (ab196495, 1:500, Abcam, USA), Bax (ab182733, 1:2000, Abcam, Cambridge, MA, USA), and β-actin (ab179467, 1:5000, Abcam, Cambridge, MA, USA). Using a Chemiluminescence Imaging System (Synoptics, Cambridge, UK) with an enhanced chemiluminescence (ECL) Western blotting substrate (Advansta Inc., Menlo Park, CA, USA), the immunoreactive bands were seen and analyzed.