Figure 1. Isolation of interacting proteins for the catalytic domain of Rho-kinase.

(A) Domain structure of Rho-kinase and the constructs used for affinity column chromatography. (B) Strategy for isolation of protein kinase substrates. (C, D) Isolation of Rho-kinase-cat-interacting proteins from rat brain cytosol (C) and P2 (D) fractions. The cytosolic or P2 fraction of rat brain lysate was loaded onto a Glutathione-Sepharose column coated with either GST, GST-Rho-kinase-cat, GST-Rho-kinase-cat-KD or GST-PKN-cat. The bound proteins were eluted by addition of 1 M NaCl after washing with 50 mM NaCl. The eluates were analyze by SDS-PAGE, and visualized by silver staining. Arrowheads indicate the GST-tagged proteins used as baits.