Fig. 2

NMI is released by activated macrophages. a NMI in the supernatant (Sup) and cytoplasmic fraction of human THP1 cells (ATCC TIB-202™) were analyzed by immunoblotting post S. typhimurium stimulation, using high-mobility group box protein 1 (HMGB1), l-lactate dehydrogenase B 1 (LDHB1), and N-myc as control. β-actin in the cell and bovine serum albumin (BSA) in the supernatant were used to show equal loading. BSA was shown using commassie blue staining. b NMI in the supernatant (Sup) and cytoplasmic fraction of THP1 cells were analyzed by immunoblotting. The cells were pretreated with (+) or without (−) Brefeldin A for 8 h and stimulated with (+) or without (−) S .typhimurium for 4 h. c NMI released by mouse RAW264.7 cells (ATCC TIB-71™) (2 × 10⁶ cells ml⁻¹) was analyzed by ELISA at different time points post LPS (1 μg ml⁻¹) stimulation. Error bars indicate ± s.e.m. from three biological replicates. d Mice (n = 5 for each group) were intraperitoneally injected with 1 × 10⁴ CFU live S. typhimurium per mouse and the concentration of the NMI in the serum were determined by ELISA. Data are presented as the mean ± s.e.m. **P < 0.01. e, f NMI in the sera of mice (n = 5 for each group) was determined by ELISA 3, 6 or 24 h after intraperitoneal injection of LPS (10 mg kg⁻¹ or 20 mg kg⁻¹) or acetaminophen (APAP) (300 mg kg⁻¹). Data are presented as the mean ± s.e.m. **P < 0.01. g ELISA analysis of NMI in the serum of 24 patients and 8 healthy individuals. Patients succumbed to severe inflammation were labeled with solid cycles. Significance in d and e were tested by unpaired Student’s t-test. Significance in g was tested by Mann–Whitney U-test. **P < 0.01