2.6. NanoBiT Complementation Assay of SERP1 and NS4B for Protein–Protein Interactions

NanoLuc^® Luciferase is divided into two subunits, large BiT (LgBiT; 18 kDa) and small BiT (SmBiT; 1 kDa peptide), which are expressed as fusions in order to target proteins of interest (Promega). The entire regions of NS2B, NS4B, or SERP1 were coupled to Nano-Luc Binary Technology (NanoBiT) vectors (pBiT1.1-C (TK/LgBiT) vector, pBiT2.1-C (TK/SmBiT) vector, pBiT1.1-N (TK/LgBiT) vector, and pBiT2.1-N (TK/SmBiT) vector; Promega, Madison, WI, USA), according to the manufacturer’s instructions. The HEK-293 cells were cultured in DMEM (Sigma-Aldrich) containing 10% FBS at a density of 1 × 10⁴ cells/well in 96-well plates for 24 h. The HEK-293 cells were transfected with a 40 ng LgBiT fusion construct and a 40 ng SmBiT fusion construct using Lipofectamine 2000 (Invitrogen). Twenty-four hours after transfection, the cells were serum-starved for 30 min in 0% FBS in an Opti-MEM I medium, and the relative luminescence unit (RLU) was measured using a Nano-Glo Live Cell Assay System (Promega) and GloMax^® Discover System (Promega).