FIGURE 5.
Induction of STAT3 phosphorylation by eNampt does not depend upon nicotinamide phosphoribosyltransferase enzymatic activity. A, macrophages were cultured in nicotinamide-free or normal medium for 24 h. The cells were then incubated in the absence (Con) or presence of 100 ng/ml eNampt (eNmt) for 24 h, and cell lysates were immunoblotted for tyrosine-phosphorylated STAT3 (pY-STAT3) and total STAT3. B, macrophages were incubated for 18 h with 100 ng/ml eNampt or 1.0 or 2.5 mg/ml NMN, and cell lysates were immunoblotted for tyrosine-phosphorylated and total STAT3. C, left blot, macrophages were incubated for 18 h with 500 nm FK866 alone, 100 ng/ml eNampt alone, or 100 ng/ml eNampt + 500 nm FK866. Whole cell lysates were immunoblotted for tyrosine-phosphorylated and total STAT3. Right blot, a similar experiment was conducted, except 100 ng/ml LPS was used instead of eNampt, based on the protocol of Busso et al. (26). D, in the two experiments shown, macrophages were incubated for 18 h in the absence or presence of 100 ng/ml wild-type Nampt (WT) or the indicated Nampt mutants. Whole cell lysates were immunoblotted for tyrosine-phosphorylated and total STAT3. kcat/Km data for each mutant, expressed as the percentage of kcat/Km for wild-type Nampt, is indicated above each lane. E, macrophages were incubated for 9 h in the absence or presence 50 or 100 ng/ml wild-type Nampt or the R392A Nampt mutant. Cell culture supernatants were analyzed for the presence of IL-6 by an enzyme-linked immunosorbent assay (n = 6 per condition).