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. 2014 May;196(10):1889–1900. doi: 10.1128/JB.00039-14

FIG 6.

FIG 6

Determination of homodimerization using a mycobacterial two-hybrid assay. Resazurin microtiter assay (REMA) with M. smegmatis expressing pairs of proteins fused to fragments 1 and 2 (F[1,2]) or fragment 3 (F[3]) of mDHFR. (A) Equal amounts of the three M. smegmatis strains were seeded in two rows containing serial 2-fold dilutions of trimethoprim (TRIM) (from 500 μg/ml to 0.5 μg/ml) or moxifloxacin (MFX) (from 1 μg/ml to 0.001 μg/ml). The last column on the right contains untreated cells. (B) Percentage of resazurin turnover measured in the plate shown in panel A as a function of drug concentration. Filled lozenges, GCN4F[1,2]/GCN4F[3] combination; empty circles, EspRF[1,2]/GCN4F[3] combination; filled circles, EspRF[1,2]/EspRF[3] combination. Low TRIM susceptibility correlates with target protein-protein interaction (dimerization) as a result of proper mDHFR reconstitution. Note that all M. smegmatis strains have similar susceptibilities to MFX, confirming that the system is TRIM dependent.