0.22 m nitrocellulose membrane

To analyze A1M secretion into the cell culture medium, equal volumes of cell culture supernatants were electrophoresed on 12% Tris-glycine SDS-PAGE gels, then proteins were transferred to 0.22 µm nitrocellulose membrane (GE Healthcare, Chicago, IL, USA) and blocked with 5% w/v milk for 60 min; they were then probed for A1M antibody (Abcam, Cambridge, UK; dilution 1:2500) at 4 °C overnight, followed by incubation with horse-radish peroxidase (HRP)-conjugated goat anti-rabbit antibody (Cell Signaling Technologies, Danvers, MA, USA) at room temperature for 1 h. The antigen–antibody complex was detected by a WesternBright ECL HRP substrate (Advansta, Menlo Park, CA, USA). To ascertain equivalent protein loading in the samples, membranes were stripped and reprobed again with a mouse anti-bovine serum albumin antibody (Abcam, Cambridge, UK). To analyze HO-1 expression, cells were lysed with a radioimmunoprecipitation assay buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1% Igepal CA-630, 1% Sodium-deoxycholate, 0.1% SDS, 1 × Complete Mini Protease Inhibitor Cocktail) and analyzed by immunblot, as described above, using HO-1 antibody (Proteintech Group, Manchester M3 3WF, UK; dilution 1:8000), then reprobed with a mouse GAPDH antibody (Proteintech Group, Manchester, UK; dilution 1:10,000).

Hearts from C57Bl/6 envenomed mice were removed and digested in RIPA buffer (Sigma-Aldrich/Merck, Darmstadt, Germany). Next, protein quantification of the supernatant was performed using Lowry reagents (DC™ Protein Assay, Bio-Rad, USA). The supernatants were suspended with LDS Sample Buffer (4x) (Life Technologies, USA), boiled, resolved by SDS-polyacrylamide gel electrophoresis (10–15% gel), and transferred onto a 0.22-µm-nitrocellulose membrane (GE HealthCare, USA). The membranes were blocked with Tris-buffered saline containing 0.01% Tween 20 and 5% nonfat dry milk. The monoclonal antibodies to PKA-γ clone [EP2647Y] (Abcam, USA) and PKA (phospho T197) clone [EP2606Y] (Abcam, USA) were diluted in blocking buffer for incubation. Antibody detection was done using Enhanced Chemiluminescence Reagent (GE Healthcare, USA).

Left ventricular tissue from the genome edited animals and age- and sex-matched controls were ground under liquid nitrogen and then diluted in RotiLoad1 buffer (Roth, Germany) at a final concentration of 40 µg/µl of total protein. Tissue was lysed by incubation for 1 h at room temperature and 4 min at 80 °C. Proteins were separated by PAGE (collection gel: 4% acrylamide/bisacrylamide, 0.4 M EDTA, 0.4% SDS and 70 mM Tris, separating gel: 8% acrylamide/bisacrylamide, 30% glycerol, 0.4% SDS, 0.1 M glycin and 0.2 M Tris-base) at 230 V, 20 mM and 20 W for 48 h at 4 °C). The myosin was subsequently blotted to a 0.22 µm nitrocellulose membrane (GE, USA) for 1,5 h at 30 V in Tris/Glycin-transfer buffer with 10% methanol. For α- and ß-MyHC detection, the membrane was washed with TBS, blocked over night with 3% milk powder non- fat (Santa Cruz, USA) in TBS, incubated with the primary antibody ab50967 (abcam, USA) for 2 h at room temperature, washed in TBS, incubated with the secondary antibody anti-mouse-HRP (BioRad, Germany) for 1 h at room temperature and washed again with TBS. Antibody staining was detected using SuperSignal West Dura (Thermo Fisher Scientific, Germany) in the LAS-system (GE, USA). To discriminate between the α- and ß-MyHC, atrium samples from porcine and human tissue that express both isoforms were used.