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Fig. 1. Depletion of endogenous FKBP12 from RyR1 preparations and ELISA binding assays. (A) Western blot of the RyR1 preparation (see Materials and Methods). Lane A, 10 µg recombinant FKBP; lane B, 10 µg RyR1 preparation; lanes C-G, recombinant GST-FKBP12 at 26 nM, 52 nM, 130 nM, 260 nM and 520 nM (20 µl loaded) to test the detection limit of the anti-FKBP12 Ab. Binding of GST-FKBP12 (in solution) to RyR1 coated onto ELISA plate was measured using different agents (see Materials and Methods). (B) Binding dependence on free Ca²⁺ concentration. Data is normalised by subtracting the binding value for EGTA (<0.01 µM free Ca²⁺). Open squares indicate Ca²⁺ buffer alone and the filled squares denote Ca²⁺ + 1 mM ATP. (C) Dose response to caffeine (open circles) or ruthenium red (filled triangles). Data is normalised by subtracting the binding in buffer alone. n = 5 ± s.e.m., *P<0.05, Student's paired t test. (D) [³H]Ryanodine binding to RyR1 preparations using the various free Ca²⁺ concentrations ± ATP, caffeine or ruthenium red. White bars indicate conditions favouring high open probability of the channel and grey bars indicate conditions promoting channel closing (Meissner et al., 1988). Data was normalised by dividing the binding values with that in the presence of EGTA. n = 5 ± s.e.m., *P<0.05 compared with the caffeine value, Student's paired t test.

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