Figure 2.

CaBP1 binds to putative CaM-binding sites in the C-terminal domain of α₁1.2. A, Schematic of C-terminal domain of α₁1.2 showing regions tested for binding CaM or CaBP1 in binding assays. B, Binding of CaM and CaBP1 to multiple sites in α₁1.2. GST-tagged α₁1.2 fragments shown in A were incubated with CaBP1 from transfected cells (left) or purified CaM (right) in the presence of 2 mm Ca²⁺. Bound proteins were detected by Western blotting with specific antibodies. Integrity and levels of GST-tagged proteins were confirmed by Ponceau staining. C, Ca²⁺-dependent and -independent binding of CaBP1. Binding of CaBP1 to GST-CT5, GST-CT6, or GST-CT7 was performed as in B and compared when assay was done with 2 mm Ca²⁺ (+) or 10 mm EGTA (-). D, Competitive binding of CaM and CaBP1 to CT6. Binding of CaBP1 or CaM to GST-CT6 was performed as in B with 2 mm Ca²⁺ and compared in the presence of increasing concentrations of purified CaM (left) or CaBP1 (right).