Western blotting validation

Based on the former DIA proteomics results and the identified significant protein biomarkers, Western blot assay was further performed to validate the significance of biomarkers in PCOS. Serum samples were homogenized in RIPA buffer containing protease inhibitor and the protein concentration was measured using a BCA method. 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate the proteins, then the proteins were electrotransferred onto the nitrocellulose (NC) membranes. After blocking with non-fat mike for 1 h and washed with Tris-buffered saline containing Tween 20 (TBST), the membranes were probed with the primary antibodies against Histone H4 (H4, Affinity, dilution 1:2000), Histone H2A (H2A, Affinity, dilution 1:1000), Trem-like transcript 1 protein (TLT-1, R&D systems, 0.1 µg/mL), Peroxiredoxin-1 (PRDX1, Affinity, dilution 1:2000), Band 3 anion transport protein (SLC4A1, Affinity, dilution 1:2000), Transferrin (Affinity, dilution 1:2000) at 4 °C over-night. And subsequently followed by incubated with horseradish peroxidase-conjugated secondary antibodies for 2 h at room temperature. Transferrin was detected as an internal reference protein [12]. Protein bands were visualized by enhanced chemiluminescence reagents and the protein intensity was quantified using Image-J software. Data was expressed as mean ± standard deviation, group comparisons were processed using the two-tailed Student’s t-test, p < 0.05 was considered as statistical significance.