SHIP1 inhibits LPS-induced Ras activation and the TLR4-MyD88 combination in macrophages. (A) SHIP1 knockdown enhances LPS-induced Ras activation in macrophages. RAW264.7 macrophages were transfected with pU6-SHIP1 or control pU6 plasmid and cultured for 48 hours. The cells were treated with 100 ng/mL LPS for the indicated time periods; Ras activation was assayed as described in “Materials and methods.” The gel was stained with Coomassie blue to show equal protein loading (GST-RBD). Similar results were observed in 3 separate experiments. (B-C) SHIP1 knockdown enhances the LPS-induced TLR4-MyD88 combination in macrophages. RAW264.7 macrophages were transfected with pU6-SHIP1 or control pU6 plasmid and cultured for 48 hours. The cells were stimulated with 100 ng/mL LPS for the indicated time periods; the TLR4-MyD88 complex was precipitated with antibody against to TLR4 (C) or MyD88 (D) and detected as described in “Materials and methods.” Similar results were observed in 3 separate experiments.