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Fig. 5. JP-45 interacts with the AID domain on the I-II loop of Ca_v1.1. (A) GST-I-II loop fusion protein or the GST-AID containing domain encompassed within Ca_v1.1 residues 336-384 were incubated with His-tagged JP-45 domain-2 fusion protein. Pull-down was performed as described in Fig. 3; proteins in the void (V), last wash (LW) or bound to the glutathione resin (B) were separated on a 12.5% SDS-PAGE, transferred onto nitrocellulose and probed with affinity-purified anti-JP-45 Ab. (B) Synthetic biotinylated peptides corresponding to the AID sequence or an unrelated biotinylated peptide were used to coat neutroavidine beads, which were subsequently incubated with His-JP-45 domain 2. Proteins present in the void, last wash or bound to the beads were separated on a 12.5% SDS-PAGE, transferred onto nitrocellulose and the immunopositive band was visualized using anti-His-tag commercial Abs. (C) A His-tagged fusion protein encompassing domain 2 JP-45 was prepared as described in Materials and Methods. Although the fusion protein migrated slower in SDS-PAGE, its identity was verified by direct sequencing (not shown) and by immunoblotting with anti-His Ab. Note that treatment of the fusion protein with DTT+DEPC eliminated its immunoreactivity.

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