Mini transfer packs

Cells were lysed with RIPA Buffer (Cell Signalling Technologies) containing PhosSTOP phosphatase inhibitors and Complete mini EDTA-free protease inhibitor cocktail (both Roche). 20 μg of protein was resolved on pre-cast 4%–15% Mini-PROTEAN TGX Gels (Bio-Rad) and then transferred to a PVDF membrane using the Trans-Blot Turbo Instrument and Mini Transfer Packs (both Bio-Rad). Membranes were blocked in 5% skim-milk in Tris-buffered saline with 0.1% Tween 20 (TBS-T) and primary antibodies (Supplementary Table S7) were diluted in blocking buffer and incubated overnight. Membranes were subsequently washed 3 times for 10 min in TBST before incubation with species specific secondary antibody (Peroxidase AffiniPure, Jackson Immuno Research) diluted in TBST, for 2 h at room temperature. A further 3 × 10 min washes were then performed. Clarity ECL Western Blotting Substrate (Bio-Rad) was used to expose antibody reactivity and was applied according to the manufacturer’s guidelines. Imaging was performed using a ChemiDoc Touch instrument (Bio-Rad).

Left ventricular tissue was obtained 24 h or 5 d after multiple trauma or sham procedure and homogenized and lysed using RIPA Lysis Buffer (EMD Millipore, Darmstadt, Germany) containing complete Mini-protease inhibitor and PhosSTOP protease inhibitor cocktail (Roche). Protein concentrations were determined in homogenates using the Pierce^® BCA Protein Assay Kit (Thermo Fischer Scientific, Waltham, MA, USA). Samples were loaded under reducing conditions onto a Mini-Protean®TGX™ Precast Gel (Bio-Rad Laboratories, Munich, Germany). After electrophoresis, the proteins were transferred by a Trans-Blot Turbo Transfer System using Mini Transfer Packs (Bio-Rad). The blots were blocked with 5% milk (C5aR1) or 5% bovine serum albumin (connexin 43 (Cx43)) in tris-buffered saline for 1 h at room temperature (RT) and incubated with antibodies (see below) overnight at 4°C. For analysis of the rat heart homogenates, rabbit anti-C5aR1 (Proteintech, Manchester, UK) and for Cx43 rabbit anti-connexin (Cell Signaling Technology, Danvers, MA) were used. HRP-conjugated anti-rabbit immunoglobulin G (IgG) (Cell Signaling) was used as a secondary antibody. Chemiluminescent HRP Hy Glo™ (Denville Scientific Inc, South Plainfield, NJ) was used. The blots were analyzed by ChemiDoc (BioRad Laboratories GmbH, Munich, Germany) and Image Lab Software (Version 5.2, BioRad).

Left ventricular tissue was obtained 72 h after multiple trauma or sham procedure and homogenized and lysed by using 1x RIPA Lysis Buffer (EMD Millipore) containing complete Mini-protease inhibitor and PhosSTOP protease inhibitor cocktail (Roche). Protein concentrations were determined in homogenates using Pierce® BCA Protein Assay Kit (Thermo Scientific). The samples were loaded under reducing conditions onto a 4–20% Mini-Protean®TGX™ Precast Gels (Bio-Rad Laboratories). After electrophoresis proteins were transferred by a Trans-Blot Turbo Transfer System using Mini Transfer Packs (both from Bio-Rad). The blots were blocked with 5% milk in tris-buffered saline (TBS) for 1 hour at room temperature (RT) and then incubated with antibodies (see below) overnight at 4 °C. For analysis of the pig heart homogenates rabbit anti-C5aR1 (Proteintech Group, Rosemont, IL, USA) and rabbit anti-Cx43 (Cell Signalling, Danvers, MA, USA) were used. After washing, HRP-anti-rabbit (Cell Signaling Technology, Danvers, MA, USA) was used as secondary antibody (1:15,000) at RT for 1 h followed by an additional washing step. Chemiluminescent HRP Hy Glo™ (Denville Scientific Inc, South Plainfield, NJ) was used for developing. The blots were analyzed by the ChemiDoc (BioRad Laboratories GmbH, Munich, Germany) and the Image Lab Software (Version 5.2, BioRad).