Fig. 2.

Increased NAMPT mRNA and protein expression in lung tissues and blood from pristane-exposed mice with pulmonary vasculitis/hemorrhage. A. The level of Nampt mRNA expression in pristane-exposed murine lung tissues was examined by RT-PCR. These studies revealed significantly increased Nampt mRNA expression (∼3-fold increase) compared to control, pristane-unexposed mouse lungs. B. Day 10 plasma levels of eNAMPT from IgG- and pristane-exposed mice (n = 9 samples/group). A 3-fold increase in eNAMPT levels was observed in pristane-exposed, IgG-treated mice (1.55± 0.3 ng/mL, n = 9) compared to non-pristane-exposed controls (1.01 ± 0.2 ng/mL, n = 9, p < 0.05). Mice receiving the ALT-100 eNAMPT mAb on Day 1 (1 mg/kg) demonstrated significantly reduced plasma eNAMPT levels. C. NAMPT protein expression in pristane-exposed lung tissues was assessed by IHC on Day 10 (Bethyl Ab, 1:1000 dilution). These studies demonstrated significant increases in NAMPT lung tissue protein expression with steady increases in NAMPT expression from Day 1 through Days 7 and 10. D/E. NAMPT protein expression in pristane-exposed lung homogenates, assessed via western blotting (Bethyl Ab, 1:1000 dilution), showed NAMPT lung tissue expression is markedly increased by pristane challenge and attenuated in mice receiving the eNAMPT-neutralizing ALT-100 mAb. Densitometric analysis was performed using Bio-Rad Image Lab 6.01 software to quantify protein expression normalized to b-actin expression.