Ecl plus luminous kit

Each of 10 μg cumulus cells protein samples were subjected to polyacrylamide gel electrophoresis, and were transferred onto a PVDF membrane (IPVH00010, Millipore, Massachusetts, USA). Further, 5% (w/v) skimmed milk was used to block the above membrane at 37°C for 2 h, and the primary antibodies (CD5L, ab45408, Abcam; ACPP, 60176-1-Ig, Proteintech Group, Chicago, USA; β-Actin, 66009-1-Ig, Proteintech Group) were respectively added for incubation at 4°C overnight. Secondary antibodies as goat anti-mouse IgG-HRP (BK0023, BEST, Xian, China) and goat anti-rabbit IgG-HRP (BK0027, BEST) were then incubated with membrane at room temperature for 1.5 h. The blots were visualized using the ECL Plus Luminous Kit (S17851, Yeasen, Shanghai, China). At last, the results were measured with Image J software.

We lysed endometrial tissues with 200 μ L of RIPA lysate (P0013B, Beyotime, Shanghai, China) plus 1 mM PMSF at 4  °C for 30 min, and then harvested the supernatant with centrifugation at 11,000 g for 10 min. The harvested protein concentrations were measured with a BCA quantitative kit (P0009, Beyotime). Samples were subjected to polyacrylamide gel electrophoresis, and were transferred onto a PVDF membrane (IPVH00010, Millipore, Massachusetts, USA). The membrane was blocked by 5% skimmed milk powder solution at room temperature for 2 h and was incubated with primary antibodies, including antithrombin III (rabbit monoclonal, ab126598, abcam, Cambridge, United Kingdom), cortisol binding globulin (rabbit monoclonal, ab110648, abcam), fetuin-A (alpha-2-HS-glycoprotein, rabbit monoclonal, ab137125, abcam), GAPDH (mouse monoclonal, 60004-1-Ig, proteintech, Beijing, China), and alpha tubulin (rabbit polyclonal, 11224-1-AP, proteintech) at 4 °C overnight. Secondary antibodies as goat anti-mouse IgG-HRP (BK0023, BEST, Xian, China) and goat anti-rabbit IgG-HRP (BK0027, BEST) were then incubated with membrane at room temperature for 1.5 h. The blots were visualized using the ECL Plus Luminous Kit (S17851, Yeasen, Shanghai, China). At last, the results were measured with Image J software.

Tissues or cells were lysed with 200 μl of RIPA lysate (BR0002, BEST, Xian, China) plus 1 mM PMSF at 4°C for 30 min. After centrifugation at 12,000 rpm for 10 min at 4°C, the supernatant was harvested and stored at -80°C. The BCA method was used to determine protein concentration using the BCA quantitative kit (P0012, Beyotime, Shanghai, China). Protein samples were subjected to polyacrylamide gel electrophoresis, which were then transferred onto PVDF membrane (IPVH00010, Millipore, Massachusetts, USA). The membrane was blocked by 5% skimmed milk powder at room temperature for 2 h. Then, the membrane was incubated with primary antibodies including Ki67 (ab92742, Abcam), proliferating cell nuclear antigen (PCNA; 10205-2-AP, ProteinTech, Chicago, USA), β-actin (ab8227, Abcam, Cambridge, United Kingdom), matrix metalloproteinase- (MMP-) 2 (10373-2-AP, ProteinTech), MMP9 (ab38898, Abcam), and TNFRSF1A (ab90463, Abcam) at 4°C overnight, followed by goat anti-rabbit IgG-HRP (BK0027, BEST) and goat anti-mouse anti-rabbit IgG-HRP (BK0023, BEST) secondary antibodies at room temperature for 1.5 h. The blots were visualized using the ECL Plus Luminous Kit (S17851, Yeasen, Shanghai, China).