Western Blotting of Testis Proteins

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.