IKKβ inhibits β‐catenin–Runt‐related transcription factor 2 (Runx2) signaling through its kinase‐independent function. A, Representative Western blots and densitometric analysis of active β‐catenin/GAPDH and Runx2/GAPDH expression in vascular smooth muscle cells (VSMCs) isolated from wild‐type (WT), kinase dead (KD), IKKβ knockout (KO), and kinase active IKKβ (KA) mice. WT samples used in Western blot were from littermate of KO mouse. Bars represent the mean±SD (1‐way ANOVA, n=3). B, Representative image of 3 independent ubiquitination assays in WT and KD VSMCs, in which β‐catenin was precipitated and blotted with antiubiquitin. C, Representative Western blots and densitometric analysis of Runx2/GAPDH expression in KO VSMCs transfected with IKKβ‐KD and IKKβ‐KA plasmids. KO VSMCs were harvested after 2 days of transfection. Control KO VSMCs were transfected with no plasmid, but transfection reagent only. Bars represent the mean±SD (1‐way ANOVA, n=5). *P<0.05, **P<0.01, ***P<0.001.