Fig. 2. Temporal and spatial distribution of actin isoforms during ES cell differentiation. (A) EBs fixed at different time points (6, 8, 12 and 15 days) were triple-stained with antibodies against all three anti-actin isoforms (-SMA, green; -SKA, blue; -CAA, red). In overlay images (d, h, l and p), beating areas are highlighted by a dashed white line. Bar, 200 µm. (B) Overlay image of a magnified beating area at day 8 co-stained with -SMA (green), -SKA (blue) and -CAA (red) antibodies. Inset, magnified part of the beating area. Bar, 20 µm. Images were acquired with a confocal microscope using either 10x (A) or 63x oil immersion objectives (B). (C) Whole-cell extracts of undifferentiated ES cell (D0) and EBs at days 6, 7, 8, 12 and 15 were analyzed by SDS-PAGE and immunoblotted with anti-SMA, anti-SKA and anti-CAA antibodies. (D) Schematic representation of expression of actin isoform during EB differentiation. Width of each bar correlates with the qualitative changes of the three actins over time.