Limd1 protein expression and localization in calvarial osteoblasts.
(A–C) Primary calvarial osteoblasts were cultured in osteogenic conditions for 3, 6, 9, or 12 days, lysed, and equal amounts of protein run on SDS-PAGE then Western blotted for the presence of the Ajuba-LIM family members Limd1 (A), WTIP (B) and Ajuba (C). Actin was used as a loading control. Primary (D) or immortalized (E) Wt or Limd1−/− calvarial osteoblasts were fixed, permeabilized and stained with Limd1 or vinculin antisera. (F) Immortalized wt or Limd1−/−calvarial osteoblasts transduced with RFP or RFP-Limd1, as indicated.