Figure 8.

The endogenous overexpression system for expression of a MFS superfamily protein. MDR1 with a C-terminal GFP tag was cloned into pDS1874 and overexpressed in DSY4684, the strain was designated as ANY-MDR1-GFP. (A) Confocal microscopy images of DSY4684, ANY-MDR1-GFP and its mutant. (B) Immunoblot analyses of their PM fractions (50 μg total protein) with anti-GFP monoclonal antibody. (C) Agar drug-diffusion assays of DSY4684 and ANY-MDR1-GFP cells. Susceptibility to the following xenobiotics were tested: Fluconazole (FLC; 10 μg), Anisomycin (ANI; 6 μg) and 4-nitroquinoline 1-oxide (NQO; 1 μg). (D) Drug resistance profiles of DSY4684, ANY-MDR1-GFP and its mutant variant determined by serial dilution assays. For serial dilution assays, the following drugs were used: ANI (4 μg/ml), FLC (4 μg/ml) and NQO (0.25 μg/ml).