Transfection of HEK293T cells

Human embryonic kidney (HEK) 293T cells (293tsA1609neo) were maintained in high glucose DMEM supplemented with 10% fetal bovine serum, 2 mM glutamine, nonessential amino acids, and gentamicin (Thermo Scientific). For transient transfection, 3.0 × 10⁶ cells were plated onto 6-cm dishes and transfected 18 h later by calcium phosphate coprecipitation using 6 μg of plasmid DNA prepared by GenElute HP plasmid miniprep kit (Sigma). 9 hours post-transfection cells were transferred to serum-free medium (CD293, Thermo Scientific). Serum-free media were harvested 48 h post-transfection and cleared by centrifugation. Following media harvest cells were washed in phosphate buffered saline (PBS) and lyzed in ice-cold RIPA buffer (Sigma Aldrich) supplemented with proteinase and phosphatase inhibitors for 15 min on ice. Lysates were transferred to microtubes and cleared by centrifugation for 15 minutes at 4 °C.