Sds page gel

Cell lysates were extracted with a lysis buffer. Western blotting was performed with 30 μg of total protein after total protein concentrations were estimated by a BCA assay, and then the protein samples were mixed with 5X SDS-PAGE protein loading buffer (Boster, Wuhan, P. R. China) and boiled. The samples were transferred to a PVDF membrane (Millipore, Darmstadt, Germany) by using the wet transfer method after being separated on 10% SDS-PAGE gels (Boster, Wuhan, P. R. China). After blocking with 5% milk in TBST for 2 h, the membranes were incubated with an anti-TRPC3 (Alomone, ACC-016, Jerusalem, Israel) or anti-β-actin (CST, PA5–85271, Massachusetts, USA) antibody overnight at 4 °C. To verify the specificity, the membranes were incubated with control peptide antigen and the primary antibody. β-actin was used as an internal control. The membranes were incubated with a horseradish peroxidase-conjugated anti-rabbit antibody at room temperature for 1 h after washing with TBST. The blots were visualized by ECL Western blotting substrate (Millipore, Darmstadt, Germany) according to the manufacturer’s instructions.

Total proteins in the cells were extracted and quantified using the bicinchoninic acid method. The samples (30 μg) were then added to each line of 10% SDS-PAGE gel (Boster, Wuhan, China) to separate and then transferred to PVDF membranes (Millipore, USA). After incubation with 5% BSA, the members were incubated with primary antibodies to CHST12 (dilution 1:1000; catalog number: 15,341-1-AP; Proteintech, Wuhan, China), β-catenin (dilution 1:1000; catalog number: 17,565-1-AP; Proteintech, Wuhan, China), E-cadherin (dilution 1:1000; catalog number: 20,874-1-AP; Proteintech, Wuhan, China), N-cadherin (dilution 1:1000; catalog number: 22,018-1-AP; Proteintech, Wuhan, China), and GAPDH (dilution 2:1000; catalog number: 60,004-1-Ig; Proteintech, Wuhan, China) overnight at 4°C. After incubation with HRP-conjugated secondary antibodies for 2 h at room temperature, the signal was developed using an enhanced chemiluminescence reagent (Boster, Wuhan, China). GAPDH was used as a loading control to determine the relative expression levels of CHST12, β-catenin, E-cadherin, and N-cadherin.

The ovarian tissues were processed by cutting into small pieces and lysing with RIPA buffer (Beyotime, P0013C) using ultrasonic waves. The protein samples in SDS-OAGE loading buffer (G2075, Servicebio) were separated through electrophoresis on a 10% SDS-PAGE gel (Boster, USA) and transferred to a PVDF membrane (Millipore, USA). Primary antibodies, including anti-GAPDH (1:1000, ab181602, Abcam), anti-fragilis (1:200, ab18976, Abcam), anti-MVH (1:5000, ab27591, Abcam), anti-TNF-α (1:200, ab1793, Abcam), anti-IL-2 (1:200, ab11510, Abcam), anti-IL-4 (1:1000, 66142-1-Ig, Proteintech), anti-p16 (1:2000, ab51243, Abcam), anti-p21(1:1000, 28248-1-AP, Proteintech), and anti-p53 (1:5000, 60283-2-Ig, Proteintech), were incubated overnight after blocking with 5% skim milk for one hour. After washing with TBS-T, membranes were incubated with secondary antibodies (1:5000) for one hour at room temperature. All protein bands were imaged using the ChemiDoc XRS system (Bio-Rad, USA) and analyzed with ImageJ software. All experiments were repeated at least three times.