Figure 2.

eNAMPT directly binds with CYBB or TLR4 (A) Normal human cases alongside UC patients were employed for IB with αNAMPT and αCYBB. WCLs (whole cell lysates) were utilized for IB with αActin (upper). Representative gel images derived from five independent healthy controls and patients are shown. NAMPT mRNA and CYBB mRNA expression measured by quantitative real-time PCR (lower). Five of ten normal human and UC patients’ data are shown. Statistical significance was evaluated by the Student’s t-test coupled with the Bonferroni adjustment (*** p < 0.001) versus human normal. (B) Colon of normal, acute and chronic colitis mouse were analyzed for IB with αNAMPT and αCYBB. WCLs were used for IB with αActin. Biological replicates (n = 10) for each condition were performed. (C) BMDMs were activated with LPS (100 ng/mL) for the durations indicated (left), then primed with LPS (100 ng/mL) for 4 h before being stimulated with ATP (1 mM) for the times indicated (right). IB in supernatant (SN) with αNAMPT and WCL with αNAMPT or αActin. (D,E) BMDMs were primed with LPS and stimulated with ATP (left) or incubated with rNAMPT (1 μg/mL) for 2 h (right). BMDMs were treated with IP with αNAMPT or αHis, trailed by IB with αTLR4, αCYBB or αCYBA (D) and IB with αASC or αNLRP3 (E). WCLs were used for IB with αTLR4, αCYBB, αCYBA, αASC, αNLRP3 or αActin. (F) Titration of fluorescently labeled TLR4 or CYBB with unlabeled NAMPT, using curve fit analysis to determine Kd (219 and 896 nM). The data come from five independent experiments that yielded comparable results.