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Blots were blocked in either 5% (w/v) nonfat milk (Lab Scientific, M-0841) or 1% (w/v) BSA in 1 Tris-buffered saline with Tween (TBST) (Teknova, T1688) for 3060 min at room temperature, incubated with primary antibodies in TBST for 1 h at room temperature, washed three times with TBST for 5 min each, incubated with fluorophore-conjugated secondary antibodies for 30 min at room temperature, washed three times with TBST and imaged on an Odyssey CLx imager (LI-COR) or ChemiDoc XRS+ imager with Clarity western enhanced chemiluminescence blotting substrates (Bio-Rad)

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Mice were intraperitoneally injected with pregnant horse serum gonadotropin (10 IU, Ningbo Sanseng Pharmaceutical Co., Ltd, Zhejiang, China)