Bcatm protein assay kit

Western blotting was performed as previously described (Wu et al., 2017 (link)). Tissue proteins were prepared from in vitro 2-week PTHrP-treated rat seminiferous tubules or in vivo 3-week PTHrP-injected testes. Tissues were homogenized and lysed with RIPA buffer (Bocai Biotechnology, China) to obtain protein samples. BCATM Protein Assay Kit (Takara, Japan) was used to measure the total protein concentrations of samples. A total protein (30 μg) each sample was added to the well of PAGE gel (10% w/v acrylamide) and electrophoresed and then the separated proteins were blotted onto the nitrocellulose membranes. The membranes were blocked with 5% non-fat milk in TBST buffer for 2 h and incubated with primary antibodies against LHCGR, CYP11A1, CYP17A1, 11β-HSD1, 3β-HSD1, or β-actin (ACTB) at 4°C overnight. After that, the membranes were washed and incubated with HRP-conjugated anti-rabbit or anti-goat IgG secondary antibodies (1:2,000, Bioword, USA) for 2 h at room temperature. The band was visualized by chemiluminescence using an ECL kit (Amersham, Arlington Heights, IL). The density of target protein was normalized to ACTB, the house-keeping protein, and the density was calculated using J-Software. All the antibodies used were listed in Supplementary Table 2.

Testes were homogenized and then were lysed with radio immunoprecipitation assay (RIPA) buffer (Bocai Biotechnology, China) to obtain total proteins. The protein concentrations of samples were measured with BCA^TM Protein Assay Kit (Takara, Japan) according to the manufacturer’s instruction. Aliquot of 50 μg of proteins were electrophoresed on 10% polyacrylamide gels containing sodium dodecyl sulfate and transferred onto nitrocellulose membranes. The membranes were blocked with 5% non-fat milk in Tris-buffered saline tween-20 buffer for 1 h. Then, the membranes were incubated overnight at 4 °C with primary antibodies against the following antigens: LHCGR, STAR, CYP11A1, 3β-HSD1, CYP17A1, 11β-HSD1, FSHR, DHH, and ACTB (listed in Supplementary Table 1). The membranes were then washed and incubated with HRP-conjugated anti-rabbit or anti-goat IgG secondary antibody (1:5000, Bioword, USA) for 1 h at room temperature and washed 3 times. The immunoreactive bands were visualized by chemiluminescence using a kit (ECL, Amersham, Arlington Heights, IL). The intensity of band was analyzed with ImageJ software.