Anti rabbit mouse igg antibody

Total protein was extracted from the uterus and the PECs using radioimmunoprecipitation assay lysis buffer supplemented with phenylmethanesulfonyl fluoride (Beyotime Biotechnology, Shanghai, China) according to the manufacturer’s instructions and detected by the Bicinchoninic Acid Protein Assay Kit (Tiangen Biotech Co., Ltd., Beijing, China). Approximately 50 μg of protein was separated by electrophoresis on polyacrylamide gels and subsequently transferred to nitrocellulose membranes. The membranes were blocked with 5% skim milk powder for 1.5 h and incubated overnight at 4 °C with the following primary antibodies: anti-ER-α (1:1000; ab3575, Abcam, Shanghai, China), anti- ER-β (1:1000; ab3576, Abcam), anti-PR (1:200; sc-539, Santa Cruz Biotechnology, Shanghai, China), anti-GAPDH (1:5000; Abcam) and anti-actin (1:5000; Abcam). After incubation, the membranes were washed three times with TBST and incubated with anti-rabbit/mouse IgG antibody (1:2000; CWBIO, Beijing, China) for 2 h at 37 °C, followed by washing with TBST. Finally, the membranes were immersed in a high-sensitivity luminescence reagent (BeyoECL Plus; Beyotime), exposed to film using a FusionCapt Advance FX7 (Beijing Oriental Science and Technology Development Co., Ltd., Beijing, China) and analyzed using Ipp 6.0 (Image Pro-Plus 6.0; Media Cybernetics).

Total protein was extracted from the ovarian tissue using a lysate containing phenylmethanesulfonyl fluoride [PMSF] (Beyotime, Shanghai, China) according to the manufacturer's instructions and detected using a BCA protein assay kit (Tiangen Biotech, China). The sample amount was 50–55 μg of protein per sample. Samples were separated by electrophoresis on polyacrylamide gels and subsequently transferred to nitrocellulose membranes. The membranes were incubated in 5% skimmed milk powder for 1.5 h, washed three times with Tris-buffered saline Tween-20 (TBST) (pH 7.6), and incubated with the primary antibodies [monoclonal anti-actin (1:1,000; Beyotime), rabbit antibody GH (1:300, bs-6579R; BIOSS), rabbit antibody GHR (1:300, bs-10661R; BIOSS), and monoclonal mouse antibody Hsp70 (1:300 BOSTER)] in primary antibody dilution buffer (Beyotime) at 4°C overnight. After washing with TBST, the membranes were incubated with anti-rabbit/mouse IgG antibody (1:2,000; CWBIO, Beijing, China) for 2 h at 37°C, followed by washing with TBST. Next, the membranes were immersed in a high-sensitivity luminescence reagent (BeyoECL Plus; Beyotime), exposed to film using a FusionCapt Advance FX7 (Beijing Oriental Science and Technology Development Co., Ltd., Beijing, China), and analyzed using Ipp 6.0 (Image Pro-Plus 6.0; Media Cybernetics).