Total erk1 2

Minced tissues were subjected to extract protein using RIPA solution supplemented with a protease inhibitor cocktail and protein phosphatase inhibitor cocktail. The protein concentration was quantified using a BCA protein quantitation kit (Thermo Fisher Scientific, USA). The 30 μg of proteins were separated via 10~12% SDS-PAGE and then transferred to nitrocellulose (NC) membranes. After blocking, the membranes were incubated with the following primary antibodies against GnRH (1:1000, Abcam, UK), Kisspeptin-1 (1:800, Proteintech, USA), GPR54 (1:2500, Poteintech), total-Sp1 (1:900, ABclonal, China), phospho-Sp1 (1:1200, ABclonal), total-ERK1/2 (1:1500, ABclonal), phospho-ERK1/2 (1:1500, ABclonal), MMP-13 (1:1200, Abcam), Col-X (1:1500, ABclonal), Col-Ⅱ (1:1200, Thermo Fisher Scientific) and β-tubulin (1:4000, ABclonal) overnight at 4 ˚C. Finally, the membranes were incubated with HRP-labeled secondary antibodies (1:5000, ZSGB-BIO, China). The protein band was visualized and measured with an ECL kit (Merck Millipore, USA). ImageJ software was applied for the analysis of band intensity.