Fig. 2

Effect of LPS on TTP protein levels in mouse macrophages. Mouse macrophages were treated with 100 ng/mL of LPS for 0, 0.5, 1, 2, 3, 4, 8, and 24 h. Proteins from a 10,000g supernatant (50 µg of protein per lane) were separated by10% SDS-PAGE and transferred onto nitrocellulose membranes in transfer buffer containing 0.1% SDS. The membrane was blocked with 5% nonfat dry milk in TTBS buffer and incubated with the anti-MBP-mTTP antibody (1:10,000 in blocking buffer for 1.5 h). After washed with TTBS buffer, the membrane was incubated with the affinity-purified goat anti-rabbit IgG (H + L) horseradish peroxidase conjugate (Bio-Rad, 1:10,000 in TTBS buffer for 1 h). Following washing with TTBS buffer, the membrane was incubated with Supersignal Western Blotting Detection Reagent (GE Life sciences) for 5 min before exposure to X-ray film for 30 s (Panel A) or 5 min (Panel B)