Inducible SPCA1a Expression in HEK293T Cells

A PiggyBac Cumate Switch Inducible Vector harboring the coding region of human SPCA1 isoform 1a, with a PA-tag (GVAMPGAEDDVV) and a tobacco etch virus (TEV) cleavage site at the N terminus, was introduced into human embryonic kidney (HEK) 293T cells along with the Super PiggyBac Transposase Expression Vector (System Bioscience, LLC, CA, USA). The resultant cell line inducibly expressing SPCA1a was cultured in Dulbecco’s modified Eagle’s medium supplemented with 4% inactivated fetal calf serum and incubated in a humidified incubator with 5% CO₂ at 37°C. After 2 days of incubation, expression of SPCA1a was induced with cumate (150 μg/ml) and 50 nM phorbol 12-myristate 13-acetate (PMA). Cells were incubated at 37°C for another 30 to 50 hours, harvested by centrifugation at 1000 to 2000g for 15 min, frozen in liquid N₂, and stored at −80°C before purification.