Chemicals
Malonyl-sildenafil was custom-synthesized by Leadgen Labs (Orange, CT) using desmethyl-sildenafil and malonic acid as building blocks. Desmethyl-sildenafil was purchased from Chemspace (Monmouth Junction, NJ). The sildenafil citrate (Revatio) and Linaclotide (Linzess) were extracted from pharmacy-grade pill/capsule in water, and stored as aliquots at -80°C. The doxycycline, puromycin, G418, and isosorbide mononitrate were from Sigma Chemical (St. Louis MO) and all other chemicals and reagents were from Fisher Scientific (Waltham, MA).

Western blotting

Cells were lysed by incubation in ice-cold lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.25% deoxycholate) supplemented with protease and phosphatase inhibitor cocktail (Fisher Scientific, Waltham, MA). Clarified protein extracts were quantified using a BCA protein quantification kit (Fisher Scientific, Waltham, MA). Proteins were then separated on 10% polyacrylamide minigels (BioRad, Hercules CA), transferred to nitrocellulose membranes and blocked with 5% BSA in PBS containing 0.025% Tween 20. Antibodies used for immunoblotting: β-actin (1:2000, Sigma), PKG2 (mAb E7; 1:200, Santa Cruz Biotechnology), Antibodies against phospho-VASP (Ser239) and total VASP were from Cell Signaling (1:1000; Danvers, MA). Blots were visualized on film using Pierce-ECL substrate.