Model ls6500

Equilibrium binding analyses of $\left[{}_{}{}^3\mathrm{H}\right]\text{Ry}$ to skeletal muscle RyR1 ( $0.05\mathrm{mg}/\mathrm{mL}$ ), cardiac muscle RyR2 ( $0.5\mathrm{mg}/\mathrm{mL}$ ), and brain cortical homogenates ( $0.5\mathrm{mg}/\mathrm{mL}$ ) were measured at 37°C for 3 h with constantly shaking in a binding buffer consisting of $250\text{\hspace{0.17em} mM}$ KCl, $14\text{\hspace{0.17em} mM}$ NaCl, $20\text{\hspace{0.17em} mM}$ HEPES, pH 7.4, and $2\mathrm{\mu }\mathrm{M}$ free ${\mathrm{Ca}}^{2+}$ (obtained by the addition of egtazic acid calculated using the software bound and determined (Brooks and Storey 1992 (link)). The concentrations of $\left[{}_{}{}^3\mathrm{H}\right]\text{Ry}$ (specific activity: $56.6\text{\hspace{0.17em} Ci}/\text{mmol}$ ; PerkinElmer) were used at $1\text{\hspace{0.17em} nM}$ , $2\text{\hspace{0.17em} nM}$ , and $10\text{\hspace{0.17em} nM}$ for skeletal, cardiac, and brain preparations, respectively. Nonspecific $\left[{}_{}{}^3\mathrm{H}\right]\text{Ry}$ binding was measured in the presence of a 1,000-fold of unlabeled ryanodine. Samples were harvested by rapid filtration through Whatman GF/B glass fiber filters (Whatman) using a 48-sample cell harvester (Brandel) to wash three times with $5\mathrm{mL}$ of ice-cold buffer containing (in mM) 250 KCl, 14 NaCl, 20 HEPES, and $2\mathrm{\mu }\mathrm{M}$
${\mathrm{Ca}}^{2+}$ , pH 7.4. $\left[{}_{}{}^3\mathrm{H}\right]\text{Ry}$ remained on filters was quantified using a Beckman scintillation counter (Model LS6500, Beckman). Experiments were repeated in three independent preparations with three replicates each, and the data was presented as $\text{mean}\pm \text{standard error of the mean \hspace{0.17em}}\left(\text{SEM}\right)$ . The DM concentration–response curves were fitted with a nonlinear logistic equation using Prism GraphPad software.