FIG. 1.

Cytokine-induced JNK phosphorylation and NF-κB activation is potentiated by MEKK-1 overexpression in βTC-6 cells. A: βTC-6 cells were transfected with GFP, GFP + wild-type MEKK-1, or GFP + kinase inactive mutant (mut) MEKK-1. The GFP-expressing cells were enriched by fluorescence-activated cell sorting. Two days after the transfection and cell sorting procedure, the cells were lysed, and proteins were separated by SDS gel electrophoresis and analyzed by immunoblotting with MEKK-1 and ERK antibodies. A representative blot from one out of three experiments is shown. B: βTC-6 cells transiently overexpressing GFP, GFP + wild-type MEKK-1, or GFP + mut MEKK-1 were either left untreated or treated with a mixture of cytokines (50 units/ml IL-1β, 1,000 units/ml IFN-γ, and 1,000 units/ml TNF-α) 2 days after the transfection and cell sorting procedure. After 30 min of cytokine exposure, the cells were lysed and proteins were separated by SDS gel electrophoresis and analyzed by immunoblotting with phospho-specific antibodies against JNK, ERK, or p38. Results from immunoblots were quantified by densitometry. Values of phospho-protein bands were related to those of non–phospho-specific protein bands. Data are presented as means ± SE for three individual experiments. *P < 0.05 using one-way ANOVA followed by Student's t test. C: βTC-6 cells transiently overexpressing GFP, GFP + wild-type MEKK-1, or GFP + mut MEKK-1 were either left untreated or treated with a cytokine mixture (50 units/ml IL-1β, 1,000 units/ml IFN-γ, and 1,000 units/ml TNF-α). After 30 min of cytokine exposure, the cells were lysed, and proteins were separated by SDS gel electrophoresis and analyzed by immunoblotting with p65 and IκB antibodies (top panels). Results from immunoblots as the one shown in the top panel were quantified by densitometry (bottom panel). Values of IκB bands were related to those of p65 bands. Data are presented as means ± SE for three individual experiments. *P < 0.05 using one-way ANOVA followed by Student's t test.