368 Exp. Mol. Med. Vol. 44(6), 363-368, 2012 Pharmaceuticals, Belmont, CA) for 10 min and/or with KT5823 for 1 h. Incubations were terminated by aspirating the medium, and the cells were washed repeatedly with ice-cold phosphate buffered saline to remove residual ra- dioactive phosphate. The cells were lysed with a lysis buf- fer containing 20 mM HEPES, 1% Triton X-100, 0.1 M NaCl, 50 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 1 mM NaVO4, and 10 μg/ml leupeptin (pH 7.2). After cen- trifugation (12,000 × g, 3 min), the soluble extract were precleared with 20 μl of protein A-agarose gel. The extracts were treated with a 10 μg of anti-α1c antibody (Alomone labs, Jerusalem, Israel) for 4 h and then with 20 μl of pro- tein A-agarose gel for 30 min. The immunoprecipitates were recovered by centrifugation and washed four times with a washing buffer containing 1% Triton X-100, 1% de- oxylcholate, 1% SDS, 150 mM NaCl, and 50 mM Tris (pH 8.5). The immunoprecipitates were separated on SDS-pol- yacrylamide gels. The dried gels were exposed to XAR film (Kodak, Rochester, NY). The dried gel was swelled with distilled water and transferred into NC paper. Protein bands were visualized with anti-α1c antibody, alkaline phos- phatase-labelled anti-rabbit antibody and a NBT/BCIP sub- strate kit (Bio-Rad, Hercules, CA). Data analysis For statistical analysis, Student's t-test was used to com - pare means, and a P value of less than 0.05 was consid- ered significant. Data were presented as means ± SEM. Acknowledgements We also thank Jung-A Park for her excellent technical assistance. This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (MEST) (311-2006-2- E00157). References Collins E, Bracamonte MP, Burnett JC Jr, Miller VM. Mechanism of relaxations to dendroaspis natriuretic peptide in canine coronary arteries. J Cardiovasc Pharmacol 2000; 35:614-8 Gao T, Yatani A, Dell'Acqua ML, Sako H, Green SA, Dascal N, Scott JD, Hosey MM. cAMP-dependent regulation of cardiac L-type Ca2+ channels requires membrane targeting of PKA and phosphorylation of channel subunits. Neuron 1997;19:185-96 Goldberg ND, Haddox MK, Nicol SE, Glass DB, Sanford CH, Kuehl FA Jr, Estensen R. Biologic regulation through opposing influences of cyclic GMP and cyclic AMP: the Yin Yang hypothesis. Adv Cyclic Nucleotide Res 1975;5:307-30 Kwak YG, Park SK, Kang HS, Kim JS, Chae SW, Cho KP, Yoo SE, Kim D. KR-30450, a newly synthesized benzopyran derivative, activates the cardiac ATP-sensitive K+ channel. J Pharmacol Exp Ther 1995;275:807-12 Lainchbury JG, Lisy O, Burnett JC Jr, Meyer DM, Redfield MM. Actions of a novel synthetic natriuretic peptide on hemodynamics and ventricular function in the dog. Am J Physiol Regul Integr Comp Physiol 2002;282:R993-8 Lee J, Kim SW. Dendroaspis natriuretic peptide administered intracerebroventricularly increases renal water excretion. Clin Exp Pharmacol Physiol 2002;29:195-7 Lisy O, Jougasaki M, Heublein DM, Schirger JA, Chen HH, Wennberg PW, Burnett JC. Renal actions of synthetic dendroaspis natriuretic peptide. Kidney Int 1999;56:502-8 Lisy O, Lainchbury JG, Leskinen H, Burnett JC Jr. Therapeutic actions of a new synthetic vasoactive and natriuretic peptide, dendroaspis natriuretic peptide, in experimental severe congestive heart failure. Hypertension 2001;37:1089-94 Puri TS, Gerhardstein BL, Zhao XL, Ladner MB, Hosey MM. Differential effects of subunit interactions on protein kinase A- and C-mediated phosphorylation of L-type calcium channels. Biochemistry 1997;36:9605-15 Schirger JA, Heublein DM, Chen HH, Lisy O, Jougasaki M, Wennberg PW, Burnett JC Jr. Presence of Dendroaspis natriuretic peptide-like immunoreactivity in human plasma and its increase during human heart failure. Mayo Clin Proc 1999;74:126-30 Schweitz H, Vigne P, Moinier D, Frelin C, Lazdunski M. A new member of the natriuretic peptide family is present in the venom of the green mamba (Dendroaspis angusticeps). J Biol Chem 1992;267:13928-32 Sumii K, Sperelakis N. cGMP-dependent protein kinase regulation of the L-type Ca2+ current in rat ventricular myocytes. Circ Res 1995;77:803-12 Tohse N, Nakaya H, Takeda Y, Kanno M. Cyclic GMP- mediated inhibition of L-type Ca2+ channel activity by human natriuretic peptide in rabbit heart cells. Br J Pharmacol 1995; 114:1076-82 Wennberg PW, Burnett JC Jr. Dendroaspis natriuretic peptide (DNP) is a potent relaxing agent in isolated canine epicardial coronary arteries. J Am Coll Cardiol 1997;29:305A