Anti phospho cpi 17

Then, 15 h post transfection, cells were treated with either 1 µM PMA (a PKC agonist, Sigma-Aldrich, St Louis, MO, USA) or 0.1% DMSO for 3 h, then washed in PBS, and lysed in RIPA buffer. Cleared lysates were run on 12% SDS-PAGE and blotted with anti-GFP (Santa Cruz B2, Santa Cruz, CA, USA), anti-phospho-Cpi-17 (Santa Cruz-17560, Santa Cruz, CA, USA), anti-Mlc2 (Cell Signaling #3672, Danvers, MA, USA), and anti phospho-Mlc2 (Cell Signaling #3675, Danvers, MA, USA).

All drugs used in these experiments were the highest purity available commercially. L-NAME, indomethacin, acetylcholine, chelerythrine, rauwolscine, and ML-7 hydrochloride were obtained from Sigma-Aldrich (St. Louis, MO, USA). Y-27632 was obtained from Calbiochem (La Jolla, CA, USA). Anti-PKC, anti-phospho-PKC (Pan), anti-MLC₂₀, and anti-phospho-MLC₂₀ at Ser19 were obtained from Cell Signaling Technology (Beverly, MA, USA). Anti-CPI-17 and anti-phospho-CPI-17 at Thr38 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Fura-2/AM was obtained from Molecular Probes (Eugene, OR, USA). Dulbecco’s modified Eagle’s medium, fetal bovine serum, penicillin, streptomycin, trypsin/EDTA, and glutamine were supplied by Gibco BRL (Rockville, MD, USA). All concentrations are expressed as the final molar concentration in the organ bath. indomethacin and ML-7 hydrochloride were dissolved in dimethyl sulfoxide (final organ bath concentration, <0.1%). Unless otherwise stated, all drugs were dissolved and diluted in distilled water.