Fig 2.

SubAB inhibits LPS-induced iNOS expression by RAW 264.7 cells. (A) RAW 264.7 cells (5 × 10⁴ cells/well) in a 24-well dish were grown in DMEM-1% FBS for 24 h. Cells were treated with LPS (0, 0.01, 0.1, 1, or 10 μg/ml) in the presence or absence of SubAB (0.5 μg/ml) for 24 h. Cell lysates were analyzed by immunoblotting with anti-iNOS or anti-α-tubulin antibodies as a control. Data are representative of at least three separate experiments. (B) RAW 264.7 cells (5 × 10⁴ cells/well) 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 the indicated times. Cell lysates were collected from cells and analyzed by immunoblotting as described above. Data are representative of three separate experiments. (C) RAW 264.7 cells (2 × 10⁵ cells/well) were incubated with LPS (10 μg/ml) for 6 h in the presence or absence of DTT (1 mM), tunicamycin (Tm; 1 μg/ml), MG132 (MG; 1 μM), SubAB (WT; 0.5 μg/ml), mSubAB (MT; 0.5 μg/ml), or DMSO as a control. Cell lysates were analyzed by immunoblotting with anti-iNOS, anti-α-tubulin, or anti-BiP/Grp78 antibodies as described above. Data are representative of three separate experiments. (D) RAW 264.7 cells (5 × 10⁴ cells/well) 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 4 or 24 h. Total RNA extracted from the cells was subjected to real-time PCR using specific primers for iNOS as described in Materials and Methods. iNOS expression was normalized to GAPDH (iNOS/GAPDH). Data are means ± SD of values from three separate experiments. Statistical significance: *, P < 0.01; **, P < 0.05; N.S., not significant.