Rat ventricular cardiomyocytes were incubated during 60 minutes in control, ouabain 2 µmol/L or istaroxime 10 µmol/L. Then, cells were collected and homogenized with lysis buffer. Protein was measured by the Bradford method using BSA as standard. Lysates (≈90 μg of total protein per gel line) were seeded in a 10% SDS polyacrylamide gel and transferred to polyvinylidene difluoride membranes. Blots were probed overnight with antibodies raised against oxidized CaMKII (Ox‐CaMKII, 1:1000; Millipore Corp, Billerica, MA), phospholamban (1:1000; Badrillal Leeds, UK) and Anti Bax (Bax, 1:500; Abcam, Cambridge, MA). After stripping the blots were probed with phospho‐Thr286‐CaMKII (1:1000; Abcam) phospho‐Thr17‐ phospholamban (p‐Thr17 phospholamban, 2:500; Badrilla), Bcl‐2 (Bcl‐2, 1:1000; Santa Cruz Biotechnology, Dallas, TX) and anti‐GAPDH (GAPDH, 1:10000; Santa Cruz Biotechnology) was used as loading control for normalization.
Immunoreactivity was visualized by a peroxidase‐based chemiluminescence detection kit (Amersham Biosciences, Amersham, UK) using a Chemidoc Imaging System (Bio‐Rad Laboratories, Hercules, CA). The signal intensity of the bands in the immunoblots was quantified by densitometry using Image J software.18 (link)
Immunoreactivity was visualized by a peroxidase‐based chemiluminescence detection kit (Amersham Biosciences, Amersham, UK) using a Chemidoc Imaging System (Bio‐Rad Laboratories, Hercules, CA). The signal intensity of the bands in the immunoblots was quantified by densitometry using Image J software.18 (link)