Activation of ERK1/2 by Fas ligation and its relationship with caspase-1 activation and DC maturation. (A) DCs were cultured in medium alone as a control (C) or were stimulated with anti-Fas antibody Jo-2 (J), isotype antibody (I), or LPS (L) for the indicated times. Whole-cell lysates were then electrophoresed and probed with phospho-ERK1/2, followed by an appropriate secondary antibody. (B) DCs were pretreated with different doses of PD98059 for 30 minutes, then stimulated with Jo-2, isotype antibody or LPS for 24 hours. The levels of IL-1β in the supernatants were determined using ELISA, and cells were harvested and analyzed for CD86 expression by flow cytometry. (C) DCs were pretreated with different doses of PD98059 for 30 minutes and stimulated with Jo-2 or isotype antibody for 8 hours. Cells were then stained with R123 and analyzed by FACS analysis. (D) DCs were pretreated with different doses of PD98059 for 30 minutes, then stimulated with Jo-2 for 30 minutes. ERK1/2 activation and caspase-1 activation were examined by immunoblotting of cell lysates with anti-phospho-ERK1/2 antibody or anti-caspase-1 P20 subunit antibody.