Plasmid Preparation for Nanoluciferase Assay

The preparation of the plasmids for human α3(IV), α4(IV), and α5(IV) for nanoluciferase complementation system was previously described.^{13 (link)} In brief, the SmBiT and LgBiT fragments were fused to the C-terminal of α3(IV) and α5(IV) in the pFC36K SmBiT TK-neo Flexi and pFC34K LgBiT TK-Neo Flexi vectors (Promega), respectively. α4(IV) was subcloned into pEB multi-Hygromycin vector (Wako) and fused with 3×FLAG tag. Mutants of α3(IV) and α5(IV) were generated by site-directed mutagenesis as previously described.^{10 (link),13 (link)} The plasmid for PPIF/CypD/CypF (HG14689-CM) was from Sino Biological. For stable cell lines, α3(IV)-SmBiT, α4(IV)-3×FLAG, and α5(IV)-LgBiT wild type or G1244D were subcloned into pLVSIN vector (Takara), respectively, containing hygromycin, puromycin, or blasticidin resistance genes. The reagents CsA, PSC-833, and trimethyl amine N-oxide (TMAO) were from Sigma-Aldrich. Dimethyl sulfoxide, glucose, mannitol, sorbitol, sucrose, and taurine were from Nacalai Tesque. Trehalose was from Tokyo Chemical Industry. Brefeldin A (BFA) was from calbiochem. ALV (HY-12559) was obtained from Medchem Express. FK506/Tacrolimus was from Abcam.