Fig. 4.
Calmodulin binds to mLin-2/CASK but does not affect X11α–mLin-2 interaction. A, GST and GST mLin-2/CASK (regions 1–320 and 1–294) were incubated with 0, 2, or 10 μg of calmodulin in the presence of 0.1 mmCa2+ or 1 mm EGTA (asterisk). After washing, bound calmodulin was resolved on SDS-PAGE, transferred to nitrocellulose, and detected with anti-calmodulin antibody. B, Lysates from untransfected A-172 cells were immunoprecipitated with preimmune or immune anti-mLin-2/CASK antibodies, and bound proteins were resolved on SDS-PAGE and transferred to nitrocellulose. Proteins were successively revealed with anti-mLin-2/CASK (top) and anti-calmodulin (bottom) antibodies. C, GST mLin-2/CASK (region 1–320) fusion protein immobilized on glutathione beads was incubated with Ca2+/calmodulin, and then lysate with (+X11α) or without (−X11α) myc-tagged X11α was added. Bound calmodulin and X11α was then assessed using immunoblotting.