Ab11425

The study was carried out following the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health and the Directive 2010/63/EU of the European Parliament. 6-8 weeks old male mice of strain C57BL6 were sacrificed by cervical dislocation and their hearts were harvested and snap frozen in liquid nitrogen and stored at -80 °C. Heart tissue was homogenized on a Precellys 24 and solubilized in ice-cold lysis buffer containing protease and phosphatase inhibitors. Tissue lysates were centrifuged to remove insoluble debris. For each tissue preparation produced, lysates derived from 5 mice were pooled and protein concentrations were measured by Quick Start Bradford Dye Reagent (Biorad). Solubilized heart tissue lysate was pre-cleared with Dynabeads protein G (Invitrogen) before incubation with primary antibody followed by binding to Dynabeads protein G, using either anti-KCNQ1 (10 μl SC10646, Santa Cruz), anti-CACNA1C (2 μl AC003, Alomone), anti-KCNH2 (2 μl AC062, Alomone), anti-CAV3 (2 μl ab2912, Abcam), anti-SNTA1 (2 μl ab11425, Abcam) or control IgG (1.5 μl goat IgG: SC2028, 1.5 μl rabbit IgG: SC2027, 1.5 μl mouse IgG: SC2025, Santa Cruz). After washing, bound proteins were eluted with 1× sample buffer containing 100 mM dithiothreitol (70 °C, 3 min) and separated by SDS-PAGE (4-15 % Bis-Tris gels, BioRad).