Our data indicate that cellular dysfunction is more severe in IDCM than in ISHD. and 4C. for 20?min at 50,000at 4C. The sediment was resuspended in binding buffer (10?mM Tris, 154?mM NaCl, pH 7.4). Protein content was measured by the method of Bradford using bovine IgG as standard. POLD1 Radioligand binding was performed as described earlier (Niclauss et al. 2006) using a 90?min incubation at 37C with [125I]iodocyanopindolol (ICYP; specific activity 2200?Ci/mmol, Perkin Elmer, Zaventem, Belgium) in a total volume of 250?l. Non-specific binding was defined as binding in the presence of 100?M isoproterenol (Sigma-Aldrich). All experiments were performed in duplicates in 96 well plates, and incubations were terminated by rapid vacuum filtration over Whatman GF/C using a Filtermate harvester (Perkin Elmer). Each filter was washed with approximately 10?ml Tirbanibulin Mesylate of buffer. Radioactivity adherent to the filters was quantified in a Topcount NXT (Perkin Elmer) using Microsint O scintillator (Perkin Elmer). To determine the relative amount of 1AR and 2AR, membranes were incubated with ICYP (100?pM) in the presence or absence of eight concentrations (range 10?10 to 10?3 M) of the highly selective 1AR antagonist CGP 20712A (1-[2-((3-carbamoyl-4-hydroxy) phenoxy)ethylamino]-3-[4-(1-methyl-4-trifluoromethyl-2-imidazolyl)phenoxy]-2-propanol Tirbanibulin Mesylate methanesulfonate). Components of Tirbanibulin Mesylate the -adrenergic receptor pathway Protein expression levels of G-coupled receptor kinases (GRK2 and GRK5), G-proteins (Gs and Gi) and protein phosphatase 1 (PP-1) were analyzed by one-dimensional 15% SDS-polyacrylamide gel electrophoresis (1D-PAGE) and subsequent Western blotting. Samples were applied in concentrations Tirbanibulin Mesylate which were within the linear range of detection: 20?g for GRK2, GRK5, Gs and PP-1, and 10?g for Gi. Blots were pre-incubated with 0.5% milk powder in TTBS (Tween-tris-buffered-saline: 10?mM TrisCHCl pH 7.6, 75?mM NaCl, 0.1% Tween) for 1?h at room temperature. The blots were incubated overnight at 4C with primary rabbit polyclonal antibodies (Santa Cruz) against GRK2 (dilution 1:1000; sc-562), GRK5 (dilution 1:1000; sc-565), Gs (Gs/olf; dilution 1:1000; sc-383), Gi (dilution 1:1000; Gi-1 sc-262, Gi-2 sc-7276, Gi-3 sc-262) or primary mouse polyclonal antibody against PP-1 (dilution 1:50; sc-7482, Santa Cruz). Specificity of the antibodies has been shown in previous studies (Vinge et al. 2001; Cho and Kehrl 2007) and all antibodies revealed one protein band in our Western blot analysis indicative for their specificity. Primary antibody binding was visualized using a secondary horseradish peroxidase-labeled goat-anti-rabbit/mouse antibody (dilution 1:2000; DakoCytomation) and enhanced chemiluminescence (ECL plus Western blotting detection, Amersham Biosciences). All signals were normalized to actin (dilution 1:1000; clone KJ43A; Sigma) stained on Tirbanibulin Mesylate the same blots. Myofilament protein phosphorylation Myofilament protein phosphorylation was determined using Pro-Q Diamond Phosphoprotein Stain as described previously (Zaremba et al. 2007). To preserve the endogenous phosphorylation status, frozen biopsies were homogenized in liquid nitrogen and re-suspended in 1?ml cold 10% trichloroacetic acid solution (TCA; dissolved in acetone containing 0.1% (w/v) dithiothreitol (DTT)). TCA-treated tissue pellets were homogenized in sample buffer containing 15% glycerol, 62.5?mM Tris (pH 6.8), 1% (w/v) SDS and 2% (w/v) DTT. Tissue samples were separated on gradient gels (Criterion trisCHCl 4C15% gel, BioRad) and proteins were stained for one hour with Pro-Q Diamond Phosphoprotein Stain. Fixation, washing and de-staining were performed according to the manufacturers guidelines (Molecular Probes). To assess protein content subsequently gels were stained overnight with SYPRO Ruby stain (Molecular Probes). Phosphorylation status of myofilament proteins was expressed relative to protein expression of cMyBP-C to correct for differences in sample loading. Staining was visualized.