Activation of NF-κB is essential for HIMF-induced Flk-1 expression. Cells were co-transfected with pNFκB-luc, dominant-negative mutants of NF-κB pathway and pRL-TK, with or without stimulation of HIMF protein for various periods as indicated. (6A) Dual-luciferase assay indicated that SVEC-HIMF had higher NF-κB activity than their control counterparts. (6B) Dual-luciferase assay indicated that HIMF protein increased NF-κB activity in SVEC 4–10 cells in a dose-dependent manner. (6C) Western blots indicated that HIMF (40 nmol/L) induced phosphorylation of IKK and IκBα in SVEC 4–10 cells. Transfection of SVEC 4–10 cells with dominant-negative mutants IKKα (K44A) and IKKβ (K44A), or super-repressor IκBα (S32A/S36A) abolished HIMF (40 nmol/L)-induced NF-κB activity. The figures indicate the relative density compared to control. (6D) The upregulation of Flk-1 induced by HIMF (40 nmol/L) in SVEC 4–10 cells were also attenuated by transfection of these dominant-negative mutants. The symbol (*) indicates a significant increase from SVEC 4–10 parent controls or controls treated without HIMF (P < 0.05). The symbol (#) indicates a significant decrease from SVEC 4–10 cells treated with HIMF only (P < 0.05). Triplicate experiments were performed with essentially identical results (n = 3).