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Fig. 1. Expression and FKBP12-binding properties of wtRyR3 and V2322DRyR3 channels. (A) RyR3-expression levels (upper blot) and FKBP12-expression levels (lower blot) were determined in the microsomal fraction of control, wtRyR3- and V2322DRyR3-overexpressing HEK293 cells (2 µg protein/lane and 25 µg protein/lane, respectively) by western blot analysis, using the monoclonal m34C RyR antibody (1/4000) and the polyclonal FKBP12 antibody (1/1000), respectively. (B) FKBP12 expression was detected in the microsomal fraction of wtRyR3-overexpressing cells (Micros) by western blot analysis, using the polyclonal anti-FKBP12 antibody (1/1000). Microsomes were treated with 25 µM FK506 and collected by centrifugation. The supernatants (Sn) of this fraction and the FK506-treated microsomes (Pellet) were analysed for FKBP12 expression. All lanes contained 25 µg protein. (C) Immunoblot (m34C RyR antibody; 1/3000) showing RyR3 in the microsomes of HEK293 cells (Micros; 2 µg) expressing either wtRyR3 or V2322DRyR3, the solubilised fraction (Sol; 8 µg) and the fraction retained after incubation with GST or GST-FKBP12 immobilised on glutathione-Sepharose 4B (GST or GST-FKBP12; 16 µg). Localization of RyR3 and of molecular mass markers are indicated.





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