FIG. 6.

LY294,002 inhibits NF-κB phosphorylation. HepG2 cells were preincubated in phosphate-free medium containing [³²P]orthophosphate (100 μCi/ml). Where indicated, the cells were incubated with 20 μM LY294,002 (LY) for 30 min prior to stimulation with IL-1β for 5 min (A and B) or for the indicated times (C). Cell lysis and immunoprecipitation of the phosphorylated NF-κB complex were performed as described in Materials and Methods. (A) Analysis of phosphorylated NF-κB proteins. pIκB, phosphorylated IκB. (B) Western analysis of phosphorylated p65/RelA proteins. IgG, immunoglobulin G antibody heavy chain; NL, no cell lysate. (C) Analysis of phosphorylated phospho-GSK-3α (serine 21-phosphorylated GSK-3α).