Fig. 1.
Overview of the cell-based BRET screening assay. (A) Schematic representation of the BRET assay. (B) HEK293T cells (4 × 105) were co-transfected with 3CL-flag and EYFP-linker-Rluc plasmid DNA in different proportions. After 48 h post-transfection, cells were collected for Western blot and BRET ratio analysis. (C) The Q/A-linker mutant could not be cleaved efficiently by 3CL. HEK293T cells were transfected with 3CL-flag and EYFP-linker-Rluc (or Q/A-linker mutant) plasmid DNA. After 48 h post-transfection, cells were collected for Western blot and BRET ratio analysis. (D) The G11A 3CL mutant disables cleaving activity. HEK293T cells were transfected with 3CL-flag (G11A 3CL mutant) and EYFP-linker-Rluc plasmid DNA. After 48 h post-transfection, cells were collected for Western blot and BRET ratio analysis. All the results shown are the average of three independent experiments. (*P < 0.05; **P < 0.01).