Fig 6.
NF-κB p50 is involved in SubAB-mediated inhibition of LPS-induced iNOS expression. (A) RAW 264.7 cells (5 × 104 cells/well) in a 48-well dish were grown in DMEM-1% FBS for 24 h. Mouse BMMs (5 × 104 cells/well) in a 48-well dish were grown in DMEM-1% FBS containing 20 ng/ml M-CSF for 24 h. Cells were treated with LPS (10 μg/ml) in the presence or absence of SubAB (WT; 0.5 μg/ml) or mSubAB (MT; 0.5 μg/ml) for 24 h. The accumulation of nitrite in culture supernatants was quantified by Griess assay as described in the legend to Fig. 1. Data are means ± SD of values from triplicate experiments. *, P < 0.01. (B) Wild-type (NF-κB1+/+) or NF-κB1 knockout (NF-κB1−/−) BMMs (5 × 104 cells/well) in a 48-well dish were grown in DMEM-1% FBS containing 20 ng/ml M-CSF for 24 h. Cell lysates were analyzed by immunoblotting with anti-p65, anti-p50, or anti-α-tubulin antibodies. (C) Wild-type (NF-κB1+/+) or NF-κB1 knockout (NF-κB1−/−) BMMs (5 × 104 cells/well) in a 48-well dish were grown in DMEM-1% FBS containing 20 ng/ml M-CSF for 24 h. Cells were treated with LPS (10 μg/ml) in the presence or absence of SubAB (WT; 0.5 μg/ml) or mSubAB (MT; 0.5 μg/ml) for 8 and 24 h. Cell lysates were analyzed by immunoblotting with anti-iNOS or anti-α-tubulin antibodies. iNOS expression was normalized to α-tubulin using densitometry (iNOS/tubulin) and is depicted in bar graphs. Data are means ± SD of values from triplicate experiments. *, P < 0.01.