Melatonin (MT) but not tryptophan (Trp) or Serotonin (ST) inhibited COX-2 and iNOS protein levels. (A) Time-dependent inhibition by MT. RAW 264.7 cells were treated with MT (1 mM) for 30 minutes followed by LPS (2 μg/mL) combined with IFNγ (400 U/mL) (LPS/IFNγ) for up to 8 hours, and, at indicated time points, cells were lysed and COX-2 and iNOS in the cell lysates were analyzed by Western blotting. Actin was measured to serve as control. (B) Concentration-dependent inhibition of COX-2 and iNOS proteins by MT. Cells were treated with MT at 0.1 to 1 mM for 30 minutes followed by LPS, IFNγ, or LPS/IFNγ for 8 hours. Cells were homogenized and COX-2, iNOS, and actin were measured by Western blotting. (C-D) Neither serotonin nor tryptophan inhibited COX-2 or iNOS proteins. RAW 264.7 cells were treated with ST (1 mM) or Trp (1 mM) for 30 minutes followed by LPS, or LPS/IFNγ for 8 hours. COX-2, iNOS, and actin were analyzed by Western blotting. Each panel of figures is representative of data from 3 separate experiments.