Gpr22

Tissues from left ventricular myocardium were used for protein extraction. Equal amounts (10–30 μg) of protein were loaded and separated by SDS-PAGE using 8–12% acrylamide gradients. Following electrophoresis, the separated proteins were transferred electrophoretically to a polyvinylidene difluoride (PVDF) membrane (Amersham Biosciences, Piscataway, NJ, USA). Membranes were incubated in blocking buffer (5% nonfat dry milk in T-TBS containing 0.05% Tween 20) overnight to block non-specific proteins. The membranes were incubated with primary antibodies against p38 (1:500, Abcam, Cambridge, MA, USA), phosphorylated p38 (1:500, Abcam, Cambridge, MA, USA), GPR22 (1:500, Invitrogen, Carlsbad, CA, USA), and β-actin (1:10,000, Millipore, Burlington, MA, USA) for 1 h at room temperature. Signals were detected with HRP-conjugated goat anti-mouse or goat anti-rabbit with ECL (Perkin Elmer, Waltham, MA, USA).

To detect GPR22 expression in cardiomyocytes and non-cardiomyocyte cells, ventricular myocardium from 2-day-old Sprague–Dawley rats was isolated and digested with collagenase (0.4 mg/mL) and pancreatin (0.6 mg/mL) in 116 mM NaCl, 20 mM HEPES (pH 7.35), 0.8 mM NaH₂PO₄, 5.6 mM glucose, 5.4 mM KCl, 0.8 mM MgSO₄. Cells were recovered by centrifugation and then resuspended in plating medium (80% DMEM, 20% M199, 15% fetal bovine serum (FBS), 100 U/mL of penicillin and streptomycin) and plated on gelatin pre-coated coverslips. For immunofluorescent staining, cells on coverslips were fixed with 4% paraformaldehyde and then permeated with 0.5% Triton X-100. Coverslips were then incubated with antibodies against GPR22 (1:200, Invitrogen, Carlsbad, CA, USA) and Troponin I (1:1000, Millipore, Burlington, MA, USA) at 4 °C overnight, followed by incubation with Alex488 or Alex594-conjugated goat anti-mouse or rabbit IgG (Invitrogen, Carlsbad, CA, USA). Samples were examined under a fluorescent microscope (BX53, Olympus, Tokyo, Japan) after nuclear counterstaining with DAPI.