*2.5. Western Blot Analysis*

Neutrophil whole-cell and nuclear extracts were isolated for Western blot analysis as described previously [13]. Nuclear extracts were isolated using the NE-PER kit (Pierce Biotechnology, Rockford, IL, USA). Proteins were resolved by SDS polyacrylamide gel electrophoresis (4%–20%) and electrophoretically transferred to polyvinylidene fluoride membranes (0.2 μm pore size). Immunodetection was performed using chemiluminescent detection with the Renaissance Western Blot Chemiluminescence Reagent Plus (Perkin Elmer Life Sciences Inc., Boston, MA, USA). Blots

were stripped using the Restore™ Western Blot Stripping Buffer (Pierce Biotechnology Inc., Rockford, IL, USA) as described by the manufacturer. Purified rabbit polyclonal antibodies to LC3B (L7543, Sigma-Aldrich), cleaved caspase-3 (#9661, Cell Signaling), caspase-3 (#9662, Cell Signaling), p62/SQSTM1 (NBP1-48320, Novus Biologicals), NFκB p65 (sc-109, Santa Cruz Biotechnology), Lamin B (sc-6216, Santa Cruz Biotechnology), and actin (sc-1616, Santa Cruz Biotechnology) were used in this study. Optical densities of antibody-specific bands were determined using Quantity One acquisition and analysis software (Bio-Rad, Hercules, CA, USA).
