The ventricles from 5 wt (3 female, 2 male), 6 mdx (3 female, 3 male), and 2 mdx-utr (both female) mouse hearts were used for the western blot experiments. Membrane proteins were isolated using the following protein isolation protocol carried out at 4°C. Frozen cardiac ventricles were cut into 15 μm slices at −20°C using a cryoslicer. Samples were weighed, blood was removed by washing the samples with ice cold PBS, and solution A (10 mM HEPES, pH = 7.4, 10% sucrose, 5 mM EDTA, 1 mM Dithiothreitol (DTT), 2 μg/ml aprotinin, 10 μg/μl leupeptin, 1 mM pefablock) was added (4 μl of solution A / mg of tissue). The tissues were homogenized on ice. After gentle centrifugation to remove bigger tissue pieces, samples were centrifuged at 1000 x g to separate the nuclei from the cytosol and the membrane protein fraction. In the next step the supernatant was centrifuged for 45 min at 100.000 x g to separate the cytosolic protein fraction from the membrane protein fraction. Membrane proteins were re-suspended in 200 μl of solution B (10 mM HEPES, pH = 7.4, 10 % sucrose, 2 mM EDTA, 1 mM DTT, 2 μg/ml aprotinin, 10 μg/μl leupeptin, 1 mM pefablock), and protein concentrations of the lysates were determined by spectrophotometry. The membrane protein lysates of equal protein concentrations were blotted on a 9% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for 80 to 90 min at a voltage of 150 V. Separated proteins were transferred to a nitrocellulose membrane (GE Healthcare, United Kingdom) using the wet transfer method for 50 min on ice. The membrane was blocked for 2 hours at room temperature using 5% bovine serum albumin (BSA) and incubated over night with the primary antibody (anti-Kir2.1 polyclonal antibody produced in rabbit; APC-026, Alomone Labs, 1:1000 in 2% BSA) at 4°C. On the next day, after 3 PBS washing steps, the membrane was incubated with the secondary antibody (anti-rabbit IgG, horse radish peroxidase-linked antibody; 7074, Cell Signaling Technology, 1:10000) at room temperature for 60 min. The protein bands were visualized using SuperSignal Western Pico Chemiluminescent Substrate (Thermo Scientific, USA) and ECL hyperfilms (GE Healthcare, United Kingdom). The protein band intensities were measured using ImageJ software (http://rsbweb.nih.gov/ij) for western blot analysis. To subtract background signals, the specific intensity peaks were defined, and the area under the curve was normalized to the area under the curve of the loading control protein intensities. An antibody (AS7) recognizing the β1/β2 subunit of membrane bound GS proteins was used as loading control (AS7 was generated by M. Hohenegger;38 AS7 complies with the original non-specific antiserum K521, and was raised against a peptide enclosing residues 8–23 in the sequence of the beta1/beta2 subunit). Western blot experiments were performed several times per heart lysate. The obtained values for every lysate were averaged. These averaged values (resulting in n = 5 for wt, n = 6 for mdx, and n = 2 for mdx-utr) were used for the statistical analyses, whereby only wt and mdx were compared.