These samples were then separated by 16% Tris-Tricine SDS-PAGE and analyzed by Western blotting using rabbit anti-human 2M Ab and HRP conjugated anti-rabbit secondary Ab and the blots were visualized by chemiluminescence. Detection of ESAT-6:2M complex in pleural fluid samples from tuberculosis positive individuals Pleural fluid samples were collected from individuals suffering from clinically diagnosed pleural TB. binds to 2M through the C-terminal end of ESAT-6.The C-terminus of ESAT-6 is a structurally undefined region that is not involved in CFP-10 binding, deletion of 6 amino acids from the C-terminal end of ESAT-6 (ESAT-6C) does not affect its binding to CFP-10, but the ESAT-6C:CFP-10 complex fails to interact with 2M. The C-terminal end of ESAT-6 in the ESAT-6:CFP-10 complex is free and available for interaction with 2M.(TIF) ppat.1004446.s003.tif (1.1M) GUID:?F0BA0792-97A3-49F6-A1BD-72B7153F4BC9 Figure S4: The ESAT-6:CFP-10 complex interacts with mouse 2M. Recombinant His-tagged ESAT-6:CFP-10 protein was bound to Ni-NTA agarose beads and incubated for 2 hours with 1 mg cell lysate prepared from BMC2 mouse macrophages. After extensive wash the bound complexes were eluted by boiling in 1 Laemmli buffer. The samples were resolved on a 16% Tris-Tricine SDS-PAGE and transferred onto a nitrocellulose membrane and probed with rabbit anti-2M Ab (Abcam, USA) followed by HRP conjugated anti-rabbit secondary Ab (Sigma-Aldrich, USA). Bands were visualized by addition of ECL reagent (GE Healthcare). Lane 1 is input control.(TIF) ppat.1004446.s004.tif (197K) GUID:?15B6160D-E07D-4CF3-84B5-419C5597F45D Figure S5: ESAT-6 does not interact with 2M in complex with HLA class I. PMA-differentiated THP-1 macrophage lysate was incubated with recombinant ESAT-6 or ESAT-6:CFP-10 protein. Mouse anti-human HLA-I Ab, clone HP1F7 (Santa Cruz Biotechnology) and Protein A/G agarose beads were used to pull down HLA-I chain molecules from this mixture (Lanes 5 and 6). Control immunoprecipitation was carried out without the addition of anti-HLA-I Ab (Lanes 3 and 4). The protein A/G bound protein complexes were dissociated by boiling in 1 SDS-PAGE loading dye and immunoblotted for detecting ESAT-6 (Panel A) or 2M (Panel B) using either rabbit anti-His Ab or rabbit anti-human 2M Ab respectively. About 10% of the total lysate used in the pull down assays were used as input controls (Lanes 1 and 2). The blots were visualized by chemiluminescence after incubation with anti-rabbit IgG HRP conjugate. Results are representative of three different experiments.(TIF) ppat.1004446.s005.tif (357K) GUID:?4434B290-A646-4B8C-A60E-3FEA5BFE6962 Figure S6: The recombinant ESAT-6:CFP-10 protein MJN110 complex downregulates surface expression of 2M molecules. PMA-differentiated THP-1 macrophages were treated with recombinant ESAT-6:CFP-10 complex protein for 2 hours at concentration of 7.5 and 12.5 M. Cells were washed and incubated with either PE conjugated anti-human 2M or PE mouse IgM, isotype (BD Pharmingen) control antibody. 2M MJN110 expression on cell surface was analyzed by flow cytometry. Results are representative of three independent experiments.(TIF) ppat.1004446.s006.tif (551K) GUID:?615481A3-690F-4FD4-AE88-33C364D29968 Figure S7: The ESAT-6:CFP-10 complex is not cytotoxic to THP-1 macrophages. PMA-differentiated THP-1 macrophages (2105/100 l/well into a 96-well microplate) were treated with indicated concentrations of ESAT-6:CFP-10 for 2 hours. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; Sigma-Aldrich) was added at a final concentration of 1 1 mg/ml for 4 hours after MJN110 which cells were lysed with a lysis buffer (20% SDS in 50% dimethyl formamide) and the absorbance was recorded at 590 nm as described earlier (Khan or pEGFP-C1-plasmid construct. After 20C24 hours, RNA was isolated from the transfected cells to synthesize cDNA. Specific primers were used for amplification of 2M and -actin by PCR from the synthesized cDNA. Amplified products were resolved on a 1.5% agarose gel and visualized by ethidium bromide staining. Results are representative of three different experiments.(TIF) ppat.1004446.s009.tif (403K) GUID:?5CE936D1-D3DF-4175-A1EA-20035377B7ED Figure S10: Determination of purity of the enriched Rough Endoplasmic Reticulum (RER) fraction. Equal amount of protein (15 g per lane) extracted from the enriched RER fraction and whole cell lysate prepared from HEK-293 cells were separated on a 16% Tris-Tricine SDS-PAGE gel, transferred to a nitrocellulose membrane and LCA5 antibody the membrane was immunoblotted for the presence of 2M (ER-specific marker), EEA1 (endosome-specific marker), LAMP2 (lysosome-specific marker) and GAPDH (cytosol-specific marker) using appropriate combinations of.