Quantity one acquisition and analysis software

Mouse macrophage and THP-I whole-cell extracts were isolated for western blot analysis as described previously [37 (link)]. 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). Blots were stripped using the Restore Western Blot Stripping Buffer (Pierce Biotechnology Inc., Rockford, IL) as described by the manufacturer. Purified rabbit polyclonal antibodies to phospho-NFκB p65 (Ser276, Cell Signaling), NFκB p65 (sc-109, Santa Cruz Biotechnology), iNOS (sc-650, Santa Cruz Biotechnology), and actin (sc-1616, Santa Cruz Biotechnology) were used. Optical densities of antibody-specific bands were determined using Quantity One acquisition and analysis software (Bio-Rad, Hercules, CA).

RBCs were pelleted by centrifugation (10,000 g, 10 min at 4 °C) and washed with cold PBS. RBCs were lysed with RIPA buffer containing protease and phosphatase inhibitors (Sigma-Aldrich, St. Louis, MO, USA). RBC lysates were resolved by SDS polyacrylamide gel electrophoresis (4–20%) and electrophoretically transferred to polyvinylidene fluoride membranes (0.2 μm pore size) according to manufacturer's instructions (Life Technologies, Carlsbad, CA, USA). Immunodetection was performed using chemiluminescence 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 monoclonal antibodies to β-spectrin 1 (ab 129065, Abcam, Cambridge, MA, USA) 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).