Fig. 1.

CSQ2 displays a direct interaction with RyR2. (A) GST pull-down experiments (n=3) of native RyR2 incubated with purified GST-fusion proteins as indicated. Solubilised pig cardiac SR vesicles were incubated with purified GST-fusion proteins that had been captured on glutathione beads. Beads were precipitated by centrifugation, and the presence of associated RyR2 was analysed by SDS-PAGE (4% gel) and immunoblotting using the Ab¹⁰⁹³ antibody (top). As negative control, pull-down assays were performed with GST alone. To detect the isolated GST-fusion proteins, one-tenth of pull-down samples was analysed by SDS-PAGE (12% gel) and immunoblotting using an antibody against GST (Ab^GST, bottom). An aliquot of solubilised pig cardiac SR corresponding to 1% (5 μg) of the amount processed in the pull-down assay was also included in the gels. As a negative control, pulldown assays were performed with GST alone; GST–FKBP12.6 was used as positive control for RyR2 interaction. (B) GST pull-down experiments (n=3) of recombinant human RyR2 incubated with purified GST-fusion proteins as indicated. Solubilised HEK293 microsomes expressing RyR2 (HEK micro) were incubated with purified GST-fusion proteins captured on glutathione beads, and analysed as described in A above. (C) Co-immunoprecipitation (co-IP) analyses (n=3) of recombinant human RyR2 co-expressed with CSQ2 in mammalian HEK293 cells. HA–CSQ2 was immunoprecipitated with an antibody against HA (Ab^HA) from solubilised HEK293 lysates, and the presence of associated RyR2 was analysed by SDS-PAGE (4% gel) and immunoblotting using Ab¹⁰⁹³ (top). As negative control, co-immunoprecipitation assays were performed with non-immune rabbit IgG (Non-immune). To detect isolated HA–CSQ2, one-tenth of the immunoprecipitate was analysed by SDS-PAGE (12% gel) and western blotting (WB) using an antibody against HA (Ab^HA, bottom). An aliquot of HEK293 cell lysate corresponding to 1% (20 μg) of the amount processed in the co-immunoprecipitation assay was also included in the gels.