Fig. 1. Westen blot analysis and [³H]ryanodine-binding assay on RyR1- and RyR3-expressing clones. (A) 50 µg of microsomal proteins obtained from RyR1- or RyR3-expressing clones were separated on 5% SDS-PAGE and analysed by western blot with anti-RyR1 or anti-RyR3 polyclonal antibodies. (B) 50 µg of RyR3 microsomes from RyR1- or RyR3-expressing clones were incubated for 1.5 hours at 36°C with 20 nM [³H]ryanodine in a solution containing 0.2 M KCl, 10 mM Hepes pH 7.4, 10 µM Ca²⁺. Nonspecific binding was determined by the addition of a 1000-fold excess of unlabeled ryanodine (20 µM). The amount of [³H]ryanodine bound was measured by membrane filtration on Whatman GF-B filters.