In vitro analysis of Dickkopf (DKK)‐4 gene in the Wnt/canonical pathway using human embryonic kidney (HEK)‐293 cells. (a) Whole cell lysate from HEK293 cells transfected with empty pcDNA3.1neo (Mock), pFLAG‐β‐catenin wild type (β‐catenin wild type), or pFLAG‐β‐catenin ΔN134 (β‐catenin ΔN134), analyzed by immunoblotting with anti‐β‐catenin, anti‐DKK4 (N‐term), and anti‐β‐actin antibodies. (b) Functional involvement of DKK1 and ‐4 in activation of the Wnt‐canonical pathway. HEK293 cells were cotransfected with canonical (super 8 × TOP‐FLASH, solid columns) or mutant (super 8 × FOP‐FLASH, open columns) transcription factor T‐cell factor/lymphocyte enhancer factor (TCF/LEF) luciferase reporters, and pRL‐TK. Five hours after transfection, medium was changed and rhDKK1 and ‐4 was added. rmWnt3a was added 12 h later and luciferase activity was measured 12 h after that. Bars, SD.