Peroxidase conjugated anti rabbit or anti mouse secondary antibodies

Cells were lysed in RIPA buffer (10 mM Tris-HCl pH = 8.0, 140 mM NaCl, 0.5% SDS) supplemented with a proteinase inhibitor cocktail and 1 mM PMSF. For poly-ADP-ribose detection, lysis buffer was also supplemented with PARG inhibitor (1 mM gallotannin). For protein detection, membranes were blocked with 1% BSA in PRB-Tween 20 (0.1%). For poly-ADP-ribose, membranes were blocked with 0.5% gelatin in PRB-Tween 20 (0.1%). Nitrocellulose membranes were stained with primary antibody at 4 °C overnight, then incubated with peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies (Sigma Aldrich) for at least 2 h at room temperature. Signal was developed using SuperSignal™ West Pico Chemiluminescent Substrate and acquired with ChemiDoc-IT2 (UVP, Meranco, Poznan, Poland).

Protein expression was analyzed by Western Bot. Neutrophils derived from spleen and bone marrow of naïve and septic mice were lysed to extract the proteins. Therefore, lysis buffer containing 10 mM Tris (pH 7.5), 150 mM NaCl, 0.5% Triton X-100, 0.1% SDS, and a complete protease inhibitor mixture (Roche) was used. Samples of 25 μg of protein was separated by polyacrylamide gel electrophorese and blotted onto PVDF membranes (Bio-Rad, Hercules, CA, USA) using the Trans-Blot Turbo Transfer System (Bio-Rad). The following antibodies were applied: anti-BAX (1:1000), anti-Bcl-xL (1:1000), anti-Bcl-2 (1:750), anti-FLIP (1:1000), anti-Mcl-1 (1:750), and anti-Survivin (1:750), (all from Cell Signaling Technology, Cambridge, UK); and anti-CIAP (1:2000, Abcam, Cambridge, UK). For signal detection, peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies (Sigma-Aldrich, St. Louis, MO, USA) and a chemiluminescent substrate system (SuperSignal West Pico PLUS, Thermo Fisher Scientific, Waltham, MA, USA) were used. Densitometric quantification was carried out using Image J. β-actin served as an internal loading control. For comparison, protein levels of WT were set as 100%.