The SLAM downstream molecules ERK and IFN-γ were activated by LMP1-mediated SAP suppression. (A) ERK1/2 activation on LMP1-H9 cells. The phosphorylation levels of ERK1/2 in LMP1-expressing T cells were examined by Western blotting. Dominantnegative (DN) mutants of TRAF2/5 could decrease the phosphorylation of ERK1/2 induced by LMP1. (B) ERK1/2 phosphorylation was down-regulated by reconstituting SAP. LMP1-H9 cells were infected with retrovirus for restoring SAP expression. The pRCMV-SAP could decrease the level of ERK1/2 phosphorylation. (C) LMP1 decreased the association of SAP with SLAM. The expression of LMP1 has reduced SAP expression on H9 cells that in turn decreased the level of SAP/SLAM interaction as demonstrated by immunoprecipitation. With an equal SLAM level, gene transduction of SAP by retrovirus in H9 cells could enhance the SAP molecules entrapped by SLAM. These data indicate that LMP1-induced SLAM activation was due to the reduction of SAP protein but did not affect the binding capacity of SAP/SLAM. (D) The induction of IFN-γ by LMP1. H9 cells were transfected with plasmids as indicated. LMP1 could enhance IFN-γ secretion on H9 cells, while TRAF2/5 dominant-negative mutants suppressed the LMP1-mediated IFN-γ secretion. In addition, overexpression of SAP on LMP1-H9 cells could also suppress IFN-γ induction by LMP1. Error bars representing the standard deviation of 3 independent experiments are shown in panels A and D.