Horseradish peroxidase conjugated anti mouse or anti rabbit antibodies

Minced penile tissue was homogenized as described (30 (link)). Penile homogenates (50 µg) were resolved on 4–20% Tris gels and transferred to polyvinylidene difluoride membrane. Membranes were probed with polyclonal rabbit anti-phospho (P)-vasodilator-stimulated-protein (VASP) (Ser-239) antibody (Cell Signaling Technology, #3114) at 1:500 dilution. Membranes were then stripped and probed with rabbit anti-VASP (Cell Signaling Technology, #3132) at 1:1,000 dilution (31 (link)). For TMT-switch assay, TMT-tagged eNOS, nNOS, and sGC were immunoprecipitated and Western blotted against mouse anti-TMT antibody (1:1,000, Pierce S-Nitrosylation Western Blot Kit). A separate set of penile homogenates (30 µg) was run on a gel for β-actin (Sigma-Aldrich) at 1:5,000 dilution as a loading control for S-nitrosylated proteins (26 (link)). Bands were detected by horseradish peroxidase conjugated anti-rabbit or anti-mouse antibodies (GE Healthcare, UK), and analyzed using National Institutes of Health Image software. Results were expressed relative to vehicle-treated sham rats.

Cell lysates were obtained with RIPA lysis buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% Triton, 0.5% deoxycholate, 2 mM EDTA, and 1 mM sodium orthovanadate) and centrifuged for 15 minutes at 4°C. Protein concentration was then determined using the Bio-Rad Protein Assay Kit (Bio-Rad, Hercules, CA). Following protein loading (40 μg/well), bands were separated on 8-12% gel using SDS-PAGE, transferred to nitrocellulose paper, blocked with 5% bovine serum albumin for 1 hour at room temperature, and incubated with the primary antibodies overnight at 4°C. The bands were then incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibodies (GE Healthcare, Toronto, Canada) for 1 hour at room temperature. The blots were developed using an enhanced chemiluminescence detection kit (Pierce Biotechnology, Rockford, IL). β-actin was used for loading control. Densitometry (Image J; National Institutes of Health) was used to assess the differences in the results.

Western blot analysis was performed to detect expression of Notch family members (Jagged-1, Jagged-2, DLL1, DLL3, DLL4, Notch1–4, and cleaved Notch (NICDs)) in a panel of OvCa cells. Cells were lysed with RIPA lysis buffer (50mM Tris-cl pH 7.4, 150mM NaCl, 1% NP40, 0.25% Na-deoxycholate, 1mM PMSF, 1x Roche complete mini protease inhibitor cocktail) and centrifuged for 15 minutes at 4°C. Protein concentrations were then measured using a Bio-Rad protein assay kit (Hercules, California). After loading the protein (25 μg/well), we separated bands on an 8%–10% gel using sodium dodecyl sulfate polyacrylamide gel electrophoresis. Bands were then transferred to nitrocellulose paper, blocked with 5% milk for 1 hour at room temperature, and incubated with primary antibodies against Notch1–4, Jagged-1, Jaged-2, and DLL4 (1:1000 dilution) and DLL3 (1:2000 dilution) overnight at 4°C. The samples were incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibodies (GE Healthcare, Waukesha, Wisconsin) for 1 hour at room temperature. Blots were developed using an enhanced chemiluminescence detection kit (Pierce Biotechnology, Rockford, Illinois). Actin was used as a loading control, and all experiments were performed in duplicate. Densitometry was performed using the ImageJ software program (Bethesda, Maryland) to interpret differences in Western blot analysis results.

MPG were homogenized in RIPA buffer and centrifuged at 10,000 × g for 30 minutes at 4°C, as described previously.^{25 (link)} Supernatants were loaded on 4-20% Tris HCl gels (Bio-Rad Laboratories, Hercules, CA), transferred to a polyvinylidene fluoride membrane, and incubated with the following primary antibodies overnight at 4°C: anti-phospho (P)-AKT (Ser⁴⁷³) and anti-P-phosphatase and tensin homolog (P-PTEN [Ser³⁸⁰/Thr^382/383], Cell Signaling Technology, Beverly, MA) at 1:1,000 dilutions, anti-RhoA (Santa Cruz Biotechnology Inc, Santa Cruz, CA) at 1:1,000 dilution, and anti-ROCK-1 and ROCK-2 (BD Transduction Laboratories, San Diego, CA) at 1:1,000 and 1:2,000 dilution, respectively. Membranes were then stripped and probed with anti-AKT, anti-PTEN (Cell Signaling Technology), or anti-β-tubulin (Abcam, Cambridge, MA) antibodies at 1:1,000, 1:1,000, and 1:7,000 dilutions, respectively. Bands were detected by horseradish peroxidase conjugated anti-mouse or anti-rabbit antibodies (GE Healthcare, Piscataway, NJ, USA), and quantified using NIH Image 1.45. All results were expressed relative to nondiabetic rats treated with vehicle.