αB-crystallin is up-regulated and phosphorylated during in vitro angiogenesis. (A) Tubular morphogenesis (morph.) of BCE cells cultured in a 3-D collagen (coll) matrix in the presence of 10 ng/mL FGF-2. Black bar = 100 μm. Bottom right panel shows a magnified confocal image of a tubular structure with actin filaments (red) connecting several cells, visualized by nuclear staining (Hoechst 33 342; blue). White bar=20 μm. (B,C) Western blot analyses of αB-crystallin expression (B) and the level of Ser59-phosphorylated αB-crystallin (C) in whole cell lysates of BCE cell monolayers on gelatin (proliferating cells) or 3-D collagen cultures forming tubular structures in the presence of 10 ng/mL FGF-2 (tubular morph.). β-catenin expression was used as a loading control. (D) Immunofluorescence analysis of embryoid bodies cultured in a 3-D collagen matrix in the presence of 30 ng/mL VEGF-A. αB-crystallin (green) was expressed in endothelial sprouts (visualized by α-CD31 staining, red). Bar = 100 μM. The boxed area is shown at higher magnification in the top left corner.