Cloning

Human cDNAs were purchased from either Open Biosystems or DNASU. For transient expression in mammalian cells, full-length CSQ2, Stim1, triadin, Fam20C, calumenin, calreticulin, and sarcalumenin, were cloned into pCCF with a C-terminal Flag tag. Fam20C (WT and D478A) was cloned into pCDNA3 with a C-terminal HA-tag. For recombinant expression in E. coli, CSQ2 (19–399) and Stim1 (22-201) were cloned into pET28 vector with a C-terminal 6X-His tag. For insect cell expression, Fam20C (93–584) was cloned into a modified pI-secSUMOstar vector (LifeSensors, Malvern, PA), in which the original SUMO tag was replaced by a MBP tag and a tobacco etch virus (TEV) protease site as previously described (Xiao et al., 2013). Site-directed mutagenesis was performed using QuikChange (Agilent Technologies, Santa Clara, CA). All the constructs were verified by DNA sequencing.