The ESAT-6 Protein of Mycobacterium tuberculosis Interacts with Beta-2-Microglobulin (β2M) Affecting Antigen Presentation Function of Macrophage
Figure 7
Soluble ESAT-6:CFP-10 reduces surface levels of β2M-associated HLA class I molecules.
(A) PMA-differentiated THP-1 macrophages were treated with 12.5 µM of either ESAT-6:CFP-10 or ESAT-6ΔC:CFP-10 protein for 2 hours. Cells were stained with (W6/32) mAb followed by FITC conjugated anti-mouse secondary Ab. Expression of surface β2M conjugated HLA class I molecules was studied by flow cytometry. Isotype-matched Ab was used as control. (B) Median fluorescence intensities of different experimental groups of Figure 7A were calculated and the results are shown as mean ± SD of 3 different experiments. (C) THP-1 macrophages were either left untreated (control) or treated with 12.5 µM of ESAT-6:CFP-10 or ESAT-6ΔC:CFP-10. After 2 hours, cell were harvested and lysates were prepared. Equal amount of protein from each experimental group was incubated with W6/32 mAb bound to protein A/G agarose. Isotype matched Ab was used as control. Pulled-down complexes (Lanes 5–8) were resolved on a 15% glycine SDS-PAGE and transferred onto a nitrocellulose membrane which was probed with anti-β2M Ab. About 10% of the lysate was used as input controls (Lanes 1–4, upper panel). Equal loading in the input samples was also confirmed by probing the input controls with anti-β-actin Ab (Lanes 1–4, lower panel).