Androgen Receptor Immunoblotting in Mouse Testes

Testes from WT, Inpp4b^-/- and cryptorchid Rxfp2^-/- mice were homogenized with a glass tissue grinder in ice-cold RIPA buffer supplemented with protease and phosphatase inhibitors [23 (link)]. The lysates were diluted tenfold and 20–30 μg of protein were resolved on SDS-PAGE and transferred to PVDF membrane. For immunoblotting, rabbit polyclonal primary antibody against AR (1:1000 dilution, catalog number 06–680, Millipore, Carlsbad, CA) and mouse monoclonal β-tubulin (1:5000, #05–661, Millipore) were used. Signal was visualized using ImageQuant LAS 500 imaging system (GE Healthcare Life Sciences, Marlborough, MA) and quantitative analysis was performed with ImageQuant TL software.