Fig. 7. SNAP25 and native RyR compete for snapin binding. Co-immunoprecipitation (co-IP) experiments of native RyR incubated with snapin radiolabelled and synthesised in vitro, in the presence of increasing amounts of SNAP25. RyR was first immunoprecipitated (A) with Ab²¹⁴² from solubilised skeletal muscle heavy SR (200 µg), or (B) with Ab¹⁰⁹³ from solubilised cardiac heavy SR (2 mg), and subsequently incubated with radiolabelled, TNT-expressed snapin together with increasing amounts of purified GST-SNAP25 as indicated (total GST content constant to 1 µM made up with GST only), and co-precipitated snapin was analysed by autoradiography (15% SDS-PAGE gels). An aliquot of the TNT reaction, 10% of the volume processed in co-IP, was included in the first lane of the autoradiogram. Densitometry analysis was carried out from three separate experiments for (C) skeletal muscle or (D) cardiac heavy SR, followed by normalisation against the control sample (with no GST-SNAP25 but in the presence of 1 µM GST only).