XN down-regulates the TNF-induced NF-κB activation through modification of p65 subunit of NF-κB. (A) XN inhibits TNF-induced nuclear translocation of p65 assayed by immunocytochemical analysis. KBM-5 cells were first treated with 50 μM XN for 4 hours at 37°C and then exposed to 0.1 nM TNF for 15 minutes. After cytospinning, immunocytochemical analysis was performed as described. (B) XN inhibits TNF-induced phosphorylation of p65. KBM-5 cells were either left untreated or pretreated with 50 μM XN for 4 hours at 37°C and then treated with 0.1 nM TNF for the indicated times. Cell extracts were prepared and analyzed by Western blotting with phosphospecific p65 antibodies. Cell extracts blotted with an anti–β-actin antibody were used as loading controls. (C) XN directly inhibits p65 binding to DNA. Nuclear extracts (NE) were prepared from KBM-5 cells treated with 0.1 nM TNF for 30 minutes, incubated with XN at indicated concentrations for 30 minutes, and EMSA was performed. (D) Reversal of XN-induced suppression of DNA binding in whole cells by DTT. Nuclear extracts were prepared from untreated KBM-5 cells or cells treated with 0.1 nM TNF for 30 minutes, incubated with 50 μM XN for 30 minutes in the presence or absence of 100 μM DTT, and then assayed for NF-κB binding to DNA by EMSA. (E) DTT reverses XN-induced suppression of DNA binding of recombinant p65 and inhibits XN-mediated suppression of recombinant p65 in vitro in A293 cells. Nuclear extracts from A293 cells transfected with p65 plasmid were incubated with 50 μM XN with or without 100 μM DTT for 30 minutes and then assayed for NF-κB binding to DNA by EMSA. (F) XN has no effect on DNA binding of p65 mutated at Cys-38 position. A293 cells were transfected with wild-type p65 and mutated p65C38S in vitro nuclear extracts were prepared and EMSA was performed.