Anp antibody

PP2A and rab7 antibodies were purchased from Cell Signaling (Danvers, USA). NKA α antibody, HPRT antibody, ANP antibody, 3-nitrotyrosine antibody, tubulin antibody, goat anti-rabbit and goat anti-mouse secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, USA). NKA α1 antibody (464.6, ab7671) was purchased from Abcam (Cambridge, UK). Alexa Fluor 568 conjugated goat anti-rabbit IgG (H + L), Alexa Fluor 488 conjugated goat anti-mouse IgG (H + L), FITC-conjugated wheat germ agglutinin were from Invitrogen Corporation (Carlsbad, USA). The McK1 antibody was a generous gift from Dr. K. Sweadner (Massachusetts General Hospital, MA). SensoLyte® FDP Protein phosphatase assay kit was from AnaSpec, Inc (AnaSpec, Inc, Japan). Primers for qPCR were from Integrated DNA Technologies. Collagenase (type I) was from Worthington Biochemical. DMEM medium, DMEM/F12 medium and fetal bovine serum were from HyClone. All other reagents were purchased from Sigma Chemical Company (St. Louis, USA).

Rat hearts were homogenized in a lysis buffer containing 20 mmol/L Tris–HCl (pH 8.0), 100 mmol/L NaCl, 1 mmol/L EDTA, 10 % NP-40 (vol/vol) and a protease inhibitor cocktail (1:100 dilution, Sigma). Proteins from neonatal rat cardiomyocytes and H9c2 cardiomyoblasts were prepared by adding RIPA buffer (P0013B, Beyotime Biotechnology, China) supplemented with a protease inhibitor cocktail. Total protein concentrations were measured using a BCA protein assay kit (Pierce, Thermo). Equal amounts of total extracted proteins from rat hearts (100 μg) or cardiomyocytes (40 μg) were separated by SDS-polyacylamide agarose gel electrophoresis (SDS-PAGE) and transferred electrophoretically to polyvinylidene difluoride (PVDF) membranes (Millipore, USA). The blots were subjected to immunoblot analysis with the primary antibodies and then incubated with secondary HRP-conjugated IgG antibodies. The bands were visualized using enhanced chemiluminescence reagent kit (Millipore, USA). Corin antibody was a gift from Dr. Ningzheng Dong (Cyrus Tang Hematology Center, Jiangsu Institute of Hematology). The other antibodies were as follows: ANP antibody (sc-18811, Santa Cruz Biotechnology Inc.), internal control β-actin (sc-47778, Santa Cruz Biotechnology Inc.). The band intensities were quantified by image J Acquisition and Analysis Software.