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Fig. 1. Snapin interaction with the BT1B2 fragment of RyR2. (A) Schematic diagram of BT1B2 sub-overlapping fragments. (B) Protein expression of RyR2 fragments in yeast: protein extracts (50 µg per sample) of yeast Y190 transformed with plasmids as indicated were analysed by western blotting using Ab^Myc (12% SDS-PAGE gel; pGBKT7: empty vector). (C) Yeast two-hybrid liquid ß-galactosidase assay of yeast Y190 transformed with the plasmids as indicated. Mean values of ß-galactosidase units obtained from five individual colonies per sample, with normalisation against the positive control pVA3 + pTD1, are shown (pLAM5 + pTD1, negative control). (D) Snapin was tested for an interaction with BT1B2 using co-immunoprecipitation (IP) assays of proteins synthesised and radiolabelled in vitro. BT1B2 and snapin were co-expressed in the TNT system in the presence of canine pancreatic microsomal membranes (PMM), BT1B2 was immunoprecipitated by Ab^Myc, and the presence of co-precipitated snapin was analysed by autoradiography (15% SDS-PAGE gel). An aliquot of the TNT reaction (10% of the volume processed in co-IP) was included in the autoradiogram, as well as individual TNT reactions for BT1B2 and snapin to serve as molecular weight standards.

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