Western Blot Analysis of INSL3 Knockdown

Equal amounts of cellular proteins (10 or 15 μg) prepared from MJ and HH cells transduced with INSL3 shRNAs or non-target shRNA lentiviral transduction particles were separated by 4–12% SDS-PAGE gel and electro-transferred onto nitrocellulose membranes. The membranes were blocked with 5% BSA in TBST (Tris-buffered saline, 0.1% Tween 20) for 1 h at room temperature, then incubated overnight with primary antibodies at 4 °C overnight. The primary antibodies and dilutions used were listed in Table S6. After washing with TBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Protein bands were visualized using the SuperSignal West Pico Chemiluminescence Substrate kit (Thermo, Rockford, IL, USA). The equivalent loading of proteins in each well was confirmed by using pan-actin [26 (link)].