Fig. 4.

eNAPRT binds to TLR4. a Western blot analysis of p-p65 in HDs macrophages upon treatment (30 min) with rNAPRT (WT) or the G379A mutant at the concentration of 1 µg/ml (n = 5). b Confocal images showing p65 localization (green fluorescence) in human macrophages treated with rNAPRT (WT) or the G379A mutant (1 µg/ml, 30 min). Original magnification ×63, scale bar: 25 µm. c SPR measurements of TLR4 (1 μM), NAPRT (100 µM), and TLR4/NAPRT (1 μM/100 nM). The measurements were performed at 25 °C and the flux was fixed at 30 μL/min. d Western blot analysis of p-p65 in scramble (sc) or TLR4 siRNA-silenced macrophages upon treatment (30 min) with rNAPRT (1 µg/ml), rNAMPT (1 µg/ml), and LPS (2 µg/ml). Box plot on the right represents quantification obtained by ImageQuant of p-p65/p65 (n = 6 for rNAPRT and LPS, n = 4 for rNAMPT, paired t-test). e Confocal images showing p65 localization (green fluorescence) in macrophages (n = 3) transfected with a scramble control siRNA (sc) or a TLR4 siRNA (white fluorescence for TLR4 staining) and treated (30 min) with rNAPRT (1 µg/ml). Original magnification ×63, scale bar: 50 µm. f Box plots showing mRNA expression levels of IL1B and IL8 evaluated by qRT-PCR upon 15 h treatments with rNAPRT (1 µg/ml), rNAMPT (1 µg/ml), and LPS (2 µg/ml) of macrophages transfected for 72 h with a scramble (sc) control siRNA or with a TLR4 siRNA (n = 8 for rNAPRT and LPS, n = 6 for rNAMPT, paired t-test). g Western blot analysis of p-p65 in macrophages derived from TLR4^−/− (n = 7) or wt (n = 5) mice treated as in d. h Box plots showing mRNA expression levels of CCL2 evaluated by qRT-PCR in macrophages derived from TLR4^−/− (n = 6) or wt (n = 4) mice treated with rNAPRT (1 µg/ml), rNAMPT (1 µg/ml), and LPS (2 µg/ml) for 15 h, unpaired t-test. Results are reported as box plots, where the line in the box defines the median and the error bars define the minimum and maximum of all data. Source data are provided as a Source Data file