Figure 7
Analysis of coronin by 2D gel electrophoresis. Cell lysates were prepared from BMN isolated from wild-type and Rac2-/- mice. Cells were pre-incubated with 10 μM cycloheximide to block de novo protein synthesis, then either left unstimulated (unstim.), or stimulated for 15 min with CB/fMLF. A. Lysates were subjected to 2D gel electrophoresis using non-linear pH 3 - 10 strips for isoelectrical focusing followed by 10% SDS-PAGE. Immunoblot analysis of 2D gels for coronin-1A shows multiple immuno-reactive spots that progressively migrate to more acidic pH (spots 1 to 5, respectively). B. Densiometric quantification of spots in A show increased levels of more acidic coronin-1A peptides after stimulation of BMN isolated from wild-type, but not from Rac2-/- mice. C. Coronin-1A was immunoprecipitated from lysates and analyzed by immunoblot with phospho-specific antibodies. Left panel shows a 10% load control for each sample immunoblotted with anti-coronin-1A antibody. Right panel shows the immuno-reactivity detected with an anti-phospho-threonine specific antibody.