SHIP1 inhibits LPS-induced IκB-α degradation but enhances NF-κB activation in macrophages. (A-B) SHIP1 increases LPS-induced NF-κB reporter gene expression. RAW264.7 macrophages in 24-well plates were transiently cotransfected with 100 ng of pGL3.5XκB-luciferase, 10 ng of pTK-Renilla-luciferase, and 0, 100, and 200 ng of pU6-SHIP1 (A) or SHIP1 (B). Total amounts of plasmid DNA were equalized using corresponding control vector. After 48 hours (A) or 24 hours (B) of culture, the cells were stimulated with 100 ng/mL LPS for 6 hours. NF-κB luciferase activity was measured using theDual-Luciferase Reporter Assay System, normalized by Renilla luciferase activity, expressed as fold induction relative to the activity in unstimulated cells transfected with control vector. Data are shown as mean ± SD (n = 3) of one typical experiment. (C-D) SHIP1 inhibits LPS-induced IκB-α degradation in macrophages. RAW264.7 macrophages were transfected with pU6-SHIP1 or control pU6 plasmid and cultured for 48 hours (C) or transfected with SHIP1 or control pBK-CMV plasmid and cultured for 24 hours (D). The cells were stimulation with 100 ng/mL LPS for the indicated time periods. The degradation of IκB-α was detected by Western blotting. Similar results were observed in 3 separate experiments.