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Fig. 3. Identification of Ca_v1.1 domains interacting with JP-45. Ca_v1.1 domains I-II, C-terminal-distal and C-terminal-proximal His-tag fusion proteins, and subunit ß1a interact with the cytosolic domain of JP-45. (A) Immunoblot of purified His-tagged fusion proteins encompassing the different Ca_v1.1 domains (10% tricine SDS-PAGE) and ß1a (10% SDS-PAGE). Immunostaining was carried out using commercial anti-poly-His Abs followed by HR-conjugated anti-mouse IgG; bands were visualized by chemiluminescence. (B) Pull-down of His-tagged fusion proteins with domain 2 of JP-45 (aa residues 1-80). GST-JP-45 domain-2 fusion protein was bound to glutathione-Sepharose beads and incubated with the His-tagged recombinant Ca_v1.1 proteins. Proteins present in the void (V) or bound to the beads (B) were separated on a 10% tricine SDS-PAGE or 10% SDS-PAGE (for the ß1a subunit), blotted onto nitrocellulose and visualized in western blots by using anti-poly-His Ab as described above. (C) GST-JP-45 domain-2 fusion protein was bound to glutathione-Sepharose beads and incubated with solubilized light SR vescicles isolated from rabbit skeletal muscle in the presence of the indicated concentration of competing His-tagged I-II loop or C-terminal-distal His-tag fusion proteins. (D) Monoclonal anti-JP-45 Abs were used to co-immunoprecipitate the complex from solubilized light microsomal vescicles isolated from rabbit skeletal muscle in the presence of the indicated concentration of competing His-tagged I-II loop or C-terminal-distal fusion proteins. Proteins bound to the beads were separated in a 10% SDS-PAGE, transferred onto nitrocellulose and probed with anti-Ca_v1.1 Abs.

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