Fig. 4. Effect of the ß1a subunit on the interaction between JP-45 and Ca_v1.1. (A) Interaction between GST-JP-45 and His I-II loop in the presence of competing purified ß1a subunit. For the fusion-protein-protein interaction, 0.57 µM of GST-JP-45 domain 2 and 1.4 µM I-II loop fusion protein were incubated in the presence of the indicated concentration of His-tagged ß1a subunit. His-tagged I-II loop fusion proteins bound to glutathione-Sepharose beads coated with GST-JP-45 domain-2 fusion protein were separated on a 10% tricine SDS-PAGE and probed with anti-poly-His Ab as described in the legend to Fig. 3. (B) Solubilized rabbit skeletal-muscle light SR vesicles were incubated with glutathione-Sepharose beads coated with GST-JP-45 domain-2 fusion protein in the absence or presence of purified ß1a subunit. Proteins present in the void (V) and bound to the beads (B), were separated on a 10% SDS-PAGE, blotted onto nitrocellulose and probed with anti-1.1 subunit Ab. (C) Solubilized rabbit skeletal-muscle light SR vesicles were incubated with anti-JP-45 Ab followed by incubation with Sepharose-protein G beads in the absence or presence of competing purified ß1a subunit. Proteins present in the void (V) and bound to the beads (B), were separated on a 10% SDS-PAGE, blotted onto nitrocellulose and probed with anti-Ca_v1.1 Ab.