Figure 5. ESAT-6 specific interactions.

(A) THP-1 macrophages were left untreated (mock) or incubated either with 5 μg/ml CFP-10 (+rCFP10) or 5 μg/ml ESAT-6 (+rESAT-6) for 1 hour. The cells were lysed and incubated with Anti- His₆− tag mAb- Magnetic agarose beads to co-immunoprecipitate the interacting proteins with the His₆− tagged bait protein and the samples were subjected to Western blot analysis for presence of Enolase-1. A protein band corresponding to Enolase1 was observed only when the co-immunoprecipitation was performed with ESAT-6, but not with CFP-10 or when no protein was present as bait, indicating specificity of the interaction. (B) PMA- differentiated THP-1 cells were treated with rESAT6 for 1 h. Cells were then fixed, gently permeabilized and stained with anti- ESAT-6 antibody followed by FITC- labeled secondary antibody and examined under the laser-scanning confocal microscope at X 60 magnification. Cell nuclei were counterstained with DAPI (blue). The represented image is an overlay of the image obtained in the green channel (excitation 488 nm) and in the blue channel (excitation 405 nm) over the Differential Interference Contrast (DIC) image of the same field. Intracellular ESAT-6 can be clearly seen in the image.