Sc 5306

Immunoblotting of the non-infarcted region of heart homogenates was performed with antibodies in the following concentrations: phosphorylated phospholamban (phospho-PLN) (Ser16) 1:1000 (A285); phospho-PLN (Thr17) 1:1000 (#sc-17024, Santa Cruz Biotechnology, Dallas, TX); phosphorylated Akt (Ser473) (#9271, Cell Signaling Technology, Danvers, MA); Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) 1:1000 (#sc-5306, Santa Cruz Biotechnology); peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) 1:1000 (#ab54481, Abcam Cambridge, MA); SERCA2a 1:1000 (IID8F6); GAPDH 1:5000 (#2118 s, Cell Signaling Technology) [11 (link)]. Densitometry was performed using Image J version 1.39 (National Institutes of Health, Bethesda, MD). Values are shown as the fold change compared to the ratio of the protein of interest relative to GAPDH in the hearts of sham rats treated with vehicle.

The protein extraction buffer contained 100 mmol/L Tris‐HCl (pH 8.0), 0.1% sodium dodecyl sulphate, 1% Triton X‐100, 150 mmol/L NaCl and protease inhibitor cocktail (Roche). The cardiac protein extracts were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride membranes (Millipore) that were incubated with the listed antibodies: Cav1.2 (1:1000, AACC‐033, rabbit polyclonal antibody; Alomone Labs, Jerusalem, Israel), CaMKII (1:1000, sc‐5306, mouse monoclonal antibody; Santa Cruz Biotechnology, Dallas, USA), NCX (1:1000, mouse monoclonal antibody, ab2869; Abcam), SERCA2a (1:5000, sc‐376235, mouse monoclonal antibody; Santa Cruz Biotechnology, Dallas, USA) and α‐tubulin (1:10 000, sc‐5286, mouse monoclonal antibody; Santa Cruz Biotechnology, Dallas, USA). Subsequently, the membranes were incubated with anti‐mouse (sc‐2056; Santa Cruz Biotechnology, Dallas, USA or anti‐rabbit (sc‐2004; Santa Cruz Biotechnology, Dallas, USA) secondary IgG antibodies at a dilution of 1:10 000. Immunoreactive proteins were detected by enhanced chemiluminescence (GE Healthcare, Chicago, USA) and quantified using the ImageJ software.

Immunofluorescence staining was carried out as described previously^{36 (link)}. Briefly, mice were transcardially perfused with 4% paraformaldehyde (4 g/100 ml) and 4% sucrose (4 g/100 ml) in PBS, pH 7.4. Brain tissue was removed and post-fixed at 4 °C for 24 h. Amygdala slices (30 μm) were prepared using a freezing microtome. Brain sections were treated with 3% (vol/vol) normal goat serum in PBS containing 0.5% Triton X-100 for 1 h. Then, the brain sections were incubated with primary antibody at 4 °C for 48 h. Slices were incubated with secondary antibodies for 2 h and exposed to 4′,6-diamidin-2-phenylindol (DAPI, 1:10000) for 5 min as a counterstain. The sections were examined with a laser-scanning confocal microscope (LSM 510, Carl Zeiss) using an omnichrome air-cooled helium/neon laser tuned to produce beams at 488 and 594 nm.
Primary antibodies used were anti-CaMKII (dilution 1:100, sc-5306, Santa Cruz Biotechnology), anti-GAD67 (dilution 1:250, MAB5406, Millipore), anti-ET1 (dilution 1:100, sc-21625, Santa Cruz Biotechnology), anti-ETAR (dilution 1:100, sc-33536, Santa Cruz Biotechnology) and anti-ETBR (dilution 1:100, sc-33538, Santa Cruz Biotechnology). All secondary antibodies were chosen according to the primary antibodies from Invitrogen. Cells were counted and analyzed by an experimenter who was blind to the sample.