pcDNA-IP3R1 was provided by K. Mikoshiba (Okasaki, Japan) (77). The plasmid expressing Wfs1 was obtained by subcloning Wfs1 into myc-tagged pCS2 + MT using Hind III and Bam H1. Ncs1 was subcloned into C-terminal p3XFLAG-CMV between Eco R1 and Bam H1. To assess interactions between IP3R1 and Ncs1-Flag and between Wfs1-myc and Ncs1-Flag, 100 μg of proteins from each transfection reaction was mixed with 2 μl of mouse anti-Flag M2 antibody (Sigma-Aldrich) containing 1 mM PMSF and 2% BSA in DWBa buffer [100 mM NaCl, 20 mM tris, 1 mM EDTA (pH 7.4), 1% Triton X-100]. For IP3R1 and Wfs1-myc antibody, 100 μg of proteins from each transfection reaction was mixed with 2 μg of a rabbit anti-myc (Sigma-Aldrich). The mixtures were vortexed and incubated at 4°C overnight. Thirty microliters of Sepharose A and 10 μl of Sepharose G (Immunoprecipitation Starter Pack, GE Healthcare) were used per sample and prepared following supplier instruction. Sepharose suspension was added to the tubes containing antibody/protein samples and mixed on a rotating wheel at 4°C for 1 hour. The samples were then centrifuged at 2000 rpm for 1 min and washed three times with 1 ml of DWBa and two times with 1 ml of DWBb [100 mM NaCl, 20 mM tris, and 1 mM EDTA (pH 7.4)] each. The pellets obtained after the last wash were resuspended in 25 μl of Laemmli sample buffer (Bio-Rad). Last, all the samples were boiled at 95°C for 5 min and centrifuged at 13,200 rpm for 1 min. The supernatants were loaded on an SDS-PAGE gel for Western blotting and analyzed with an anti-myc antibody for Wfs1-myc and Ncs1-Flag coimmunoprecipitation, with the rabbit Rbt476 pan anti-IP3R antibody (a gift from J. Parys, Leuven, Belgium) (78) for Ncs1-Flag and IP3R1 coimmunoprecipitation and Wfs1-myc and IP3R1 coimmunoprecipitation. Thirty micrograms of protein from Ncs1-Flag and Wfs1-myc transfection reactions was used as controls.

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