Anti nr1d1

The relative expression patterns of NR1D1 and NR4A2 proteins in HPG from the adult yaks and testis tissues from animals of different ages were examined using Western blot. Total protein was extracted from 100 mg of each tissue sample using RAPI (Solarbio, Beijing, China). Protein concentration was determined using a BCA kit (Solarbio). 100 μg of total protein samples were electrophoresed in a sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) for Western blot analysis. The blots were electro-transferred onto a PVDF membrane (Millipore CAT, Billerica, MA, USA), and blocked with Tris-HCl buffer (Solarbio) containing 5 % (w/v) non-fat milk (Solarbio, Beijing, China) for 2 h at room temperature. The membranes were incubated at 4 °C overnight with rabbit monoclonal anti-NR1D1 (1:300), anti-NR4A2 (1:300), and anti-β-actin (1:4000, Bioss, Beijing, China) primary antibodies. The subsequent procedures were carried out as described previously [22 (link)]. All immunoblot assays were performed at least in triplicate. Optical densities of the bands were quantified and scanned using Image-Pro Plus 6.0 (Media Cybernetics Co., Rockville, USA). The expression level of β-Actin was used as an endogenous control. The expression patterns of NR1D1 and NR4A2 proteins in the hypothalamus (4 years old) or testis tissue (2 years old) were used as controls. Data were presented as mean ± SD.