Effects of violacein on the expression of proteins involved in the TNFR1 signaling, NF-κB p65 translocation, and TNF-α production. (A) After incubation for 24 hours with violacein, equal amounts of protein (50 μg) of total lysates were subjected to immunoblot analysis with TRAF1 and 2, TRADD, NF-κB (p50 and p65) antibodies. The profile of NF-κB (p50 and p65) was also evaluated for samples from human lymphocytes (L). (B) Nuclear extracts were subjected to immunoblot analysis with NF-κB p65 antibody. (C) Cells were treated with different concentrations of violacein for 24 hours, and the TNF-α concentrations were determined in the supernatants by ELISA. Results represent the mean value ± standard error of the mean (SEM).