Phosphatase inhibitors cocktail

PBMCs were lysed on ice for 30 min using RIPA lysis buffer (Beyotime) with protease inhibitors (Beyotime) and phosphatase inhibitors cocktail (Beyotime), and the protein concentration was determined by a bicinchoninic acid assay kit (Biosharp). The proteins (50 µg per lane) were mixed with one-fifth volume of 5× loading buffer, separated by 10% SDS-PAGE, and were then transferred onto polyvinylidene difluoride membranes (Millipore). The membranes were blocked for 1 h with 5% nonfat milk at room temperature and were incubated overnight at 4°C with primary antibodies against STAT3 rabbit mAb (79D7, Cell Signaling Technology), phospho-STAT3-Tyr₇₀₅ rabbit mAb (D3A7, Cell Signaling Technology), NF-κB p65 rabbit mAb (D14E12, Cell Signaling Technology), anti-NF-kB p65-phospho-S₅₃₆ antibody (EP2294Y, Abcam), and β-actin rabbit monoclonal antibody (Beyotime). After 3 washes with Tris-buffered saline with 0.1% Tween® 20 detergent, the membranes were incubated for 1 h at room temperature with anti-rabbit horseradish peroxidase-conjugated secondary antibody (GE) and were detected with enhanced chemiluminescence (Beyotime). The results were analyzed using Image Studio software.

The washing buffer (5 $\times$ 10^− 3 м MgCl₂ and 4.5 g L^− 1 glucose in Dulbecco’s PBS) was obtained from Sigma-Aldrich (St. Louis, MO, USA), and the binding buffer was composed with yeast tRNA (0.1 mg mL^− 1, Sigma-Aldrich) and bovine serum albumin (BSA; 1 mg mL^− 1 (ThermoFisher Scientific, Waltham, MA, USA) in washing buffer, which was used to reduce background binding. Super GelRed was purchased from US Everbright® Inc (Sayreville, NJ, USA). Beijing Labor Technology Co, Ltd. provided the Cell Counting Kit-8 (CCK-8) (Beijing, China; cat. no. 21,162,196). Protein markers were purchased from Sangon Biotech Co. Ltd. (Shanghai, China). The anti-PTK7 rabbit monoclonal antibody was purchased from Abcam (Cambridge, MA, USA). The Alexa Fluor 488-labeled goat anti-mouse secondary antibody, Hoechst 33,342, lysotracker, radioimmunoprecipitation assay (RIPA) lysis buffer, phosphatase inhibitors cocktail, protease inhibitors, and annexin V-FITC Apoptosis Detection Kit (no. 011821210207) were all purchased from Beyotime Biotech Inc (Jiangsu, China). All chemicals for synthesis and purification were purchased from Energy Chemical (Shanghai, China). All tubes and plates for cell culture were purchased from NEST Biotechnology (Jiangsu, China). Unless otherwise stated, all other bioreagents were purchased from Sigma.

Cells transiently expressing wild-type receptor or variants were stimulated with the FSHR agonist (S20150007, Jinsai, Changchun, China) for the indicated durations. Drug incubation was terminated by washing the cells with ice-cold phosphate buffered saline followed by the addition of lysis buffer containing complete protease inhibitor (P1010, Beyotime, Shanghai, China) and phosphatase inhibitors cocktail (P1082, Beyotime, Shanghai, China). Equal protein amounts from cell lysates were electrophoresed on a 10% SDS-polyacrylamide gel, and then transferred to a PVDF membrane and incubated with rabbit monoclonal anti-phospho-ERK1/2 antibody (4370, Cell Signaling, Danvers, USA) followed by stripping and reprobing with anti-total ERK antibody (4695, Cell Signaling, Danvers, USA) according to manufacturers’ protocols. Chemiluminescence was detected using an ECL substrate (FD8000, Fdbio science, Hangzhou, China) with ChemiDoc Touch Imaging System (Bio-Rad, Hercules). All immunoblots were semi-quantified using the Adobe Photoshop CC software, and ERK1/2 activation was calculated as the level of phosphor-ERK1/2 normalized by the total-ERK.