Figure 2.

Analysis of the binding of the D₃D₄ domain of ankyrin to cdb3. His-tagged cdB3 was incubated for 2h with increasing concentrations of a GST fusion construct of the D₃D₄ domain of ankyrin and then captured onto Co²⁺-NTA agarose beads. After washing 3x with binding buffer, bound protein was eluted and quantitated (A) by immunoblot analysis using an antibody to D₃D₄ ankyrin, or (B) by measuring GST activity in the eluent (see Methods). (C) Alternatively, GST- D₃D₄ was incubated for 2 hrs with either wild type or a mutant cdB3 lacking loop 2, and the complexes were captured on GST-Sepharose beads, washed 3x with binding buffer and evaluated for cdb3 content by dot blot analysis using an anti-cdB3 antibody. kdcB3 - kidney cdB3; cdB3 ΔL1 – cdB3 lacking loop 1; cdB3 Δ1–50 – cdB3 lacking loop residues 1–50. Error bars correspond to S.D., where n=2.