Fig. 3. Snapin interaction with the BT1B2 fragment of RyR1 and RyR3. (A) Protein expression of the corresponding BT1B2 fragments from the three mammalian RyRs and SNAP25 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). (B) 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, are shown. (C) Snapin was tested for an interaction with the corresponding BT1B2 fragment of RyR1 and RyR3 using co-immunoprecipitation (IP) assays of proteins synthesised and radiolabelled in vitro. BT1B2R1/R3 and snapin were co-expressed in the TNT system in the presence of canine pancreatic microsomal membranes (PMM), the RyR fragment 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 BT1B2R1/R3 and snapin.