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.