Western Analysis of Control and CN-injured Corpora Cavernosa
Western analysis assaying for SHH protein was performed on protein samples isolated from corpora cavernosa from control (n = 5) and CN-injured (n = 5) Sprague Dawley rat penes 21 days after CN injury, as outlined previously. Proteins were separated via electrophoresis using a 10% polyacrylamide gel and transferred to a nitrocellulose membrane (Bio-Rad) using a Hoefer SemiPhor Semi-Dry Transfer Unit (Amersham Pharmacia, Piscataway, NJ) for 1 h. Membranes were blocked for 1 h at room temperature in 5% nonfat skim milk in PBS Tween buffer. Membranes were incubated with either 1:50 SHH (N-19, SC-1194, Santa Cruz) or 1:50000 ACTB (formerly known as p-ACTIN, Sigma) antibodies overnight at 4°C. Membranes were washed with PBS-Tween one time for 10 min and then incubated with 1:45 000 anti-goat and 1:70 000 anti-mouse secondary antibodies for 1 h at room temperature. Protein bands were visualized using HRP-conjugated anti-biotin (ECL western blotting detection reagent, Amersham) according to manufacturer’s directions and were exposed to Hyperfilm.