Evaluating Protein Levels in Male Pelvic Ganglia

MPGs were excised and homogenized in a Tris-HCL (pH 7.5) buffer to evaluate the protein level of ROCK1 and ROCK2 by Western blot12 (link). Sol-O-Buffer (Fabgenix, Frisco, TX) was used to assay nNOS monomers and dimers in the MPG under non-reducing conditions38 (link). Protein (30 μg) was loaded on a 4–12% NuPage Bis-Tris gel (Invitrogen, Carlbad, CA) and separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Electrophoresis was done at 4 °C for nNOS monomers and dimers. Proteins were transferred to polyvinylidene fluoride membranes and incubated with primary antibodies (ROCK1, ROCK2 (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA), total casapse-3, activated caspase-3 (1:1000 Cell Signaling, Beverly, MA), nNOS (1:200 Santa Cruz), GAPDH (1:2500 BD Bioscience) and actin (1:500 (Sigma Aldrich, St. Louis, MO)) overnight at 4 °C. The membranes were incubated with a horseradish peroxidase-linked secondary antibody and visualized using an enhanced chemiluminescence kit (Amersham). The densitometry results were quantified using software Image J (National Institutes of Health) and normalized by actin level37 (link).