Figure 2.

Specificity of anti-pT445. (A) HA-α-adducin (40 fmol), containing the indicated amounts of HA-α-adducin phosphorylated by Rho-kinase, and HA-α-adducin-AA (40 fmol) phosphorylated by Rho-kinase were subjected to immunoblot analysis with anti-pT445, anti-pT445 preincubated with a 100-fold amount of antigen-phosphopeptide, and anti-HA antibody. The amount of phosphates incorporated into HA-α-adducin was simultaneously determined by [γ-³²P]ATP. (B) Anti-pT445 reacts with only α-adducin phosphorylated by Rho-kinase. HA-α-adducin, HA-β-adducin, or 6 X His-γ-adducin fragment (319–671 aa) was separately phosphorylated with GST-CAT, resolved by SDS-PAGE, and visualized by an image analyzer (right). 40 fmol (as ³²P-incorporated amount) of phosphorylated proteins was subjected to immunoblot analysis with anti-pT445 (left). From the top, arrows indicate the positions of the intact HA-α-adducin, HA-β-adducin, or 6 X His-γ-adducin, respectively. Anti-pT445 also bound to the degradation products of HA-α-adducin. The results are representative of three independent experiments.