Fig. 1. Biochemical analysis of the purified IP₃Rs. (A) SDS–PAGE of the loading sample and the FPLC fractions around the IP₃R peak. The gel was stained with Coomassie Blue. In lane 1, 5.0 µg of the loading sample was applied. Lanes 2–12 correspond to the fractions from the elution volume 6.0–15 ml. A 20 µl aliquot of each fraction was used for each lane. The protein concentration in fractions corresponding to lanes 4–6 was ∼0.1 mg/ml. Fractions corresponding to these lanes were used in all other experiments. A control run of the high molecular weight standards showed that dextran 2000 came out at 7.8 ml and ferritin (437 kDa) at 14.5 ml, which correspond to lanes 4 and 12, respectively. (B) Immunoblotting of the purified IP₃Rs with specific antibodies against the type 1, 2 and 3 IP₃Rs and an antibody against RyRs (both types 1 and 2). The positive control for the type 2 IP₃R antibody was microsomes made from mouse cardiac muscle, and that for the type 3 IP₃R antibody was made from renal cells (data not shown). The positive control (CTL) for the RyR antibody was the microsomes made from mouse skeletal muscle.