FIG. 4.

Effects of MEKK-1 d-siRNA on MEKK-1 levels and JNK, p38, ERK, or MKK4 activation in dispersed human islet cells. A: Dispersed human islet cells were treated with GL-3 or MEKK-1 d-siRNA. Two days after the d-siRNA treatment, the cells were lysed, and proteins were separated by SDS gel electrophoresis and analyzed by immunoblotting with MEKK-1 antibodies. A representative blot from one out of two experiments is shown. B: Dispersed human islet cells were transfected with GL3 d-siRNA or MEKK-1 d-siRNA and 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). After the 30-min cytokine exposure, the cells were lysed, and proteins were separated by SDS gel electrophoresis and analyzed by immunoblotting with phospho-specific JNK, ERK, and p38 antibodies. The results were quantified by densitometric scanning of three experiments and expressed as means ± SE. C: Dispersed human islet cells were treated with GL-3 or MEKK-1 d-siRNA. Two days after the d-siRNA treatment, the cells were exposed to 50 units/ml IL-1β. After 30 min of IL-1β treatment, the cells were lysed, and proteins were separated by SDS gel electrophoresis and analyzed by immunoblotting. MKK4 activation was determined by calculating the ratios between P-MKK4 intensities and ERK intensities. Values are means ± SE for three observations.