Western Blot Analysis of C3 and Acrolein

Serum (equal volumes) or protein lysates (10 µg) were heated with sample buffer (3.2% SDS, 32% glycerol, 0.16% bromophenol blue, 100 mM Tris-HCl, pH 6.8, 8% 2-mercaptoethanol). They were then electrophoresed on Criterion Stain-free 4–20% SDS-PAGE gels (Bio-Rad) and transferred onto nitrocellulose membranes at 100 volts for 30 min. The membranes were blocked with TBS containing 5% nonfat dry milk and probed with anti-C3 antibody (Santa Cruz, sc-28294, 1:1,000) or anti-acrolein antibody (NOVUS, NB200–556, 1:1,000) overnight at 4 °C. They were then washed three times in TBS containing 0.1% Tween 20 and incubated with horseradish peroxidase-conjugated secondary antibody for 2 h at room temperature. Signals were detected using enhanced chemiluminescence and Gel Doc System (Bio-Rad). The blots were stripped and probed for housekeeper proteins transferrin (serum) or β-actin (tissue lysate). The signal intensity was quantified using Image Lab (Bio-Rad) and NIH ImageJ software (v1.45 s).