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. 2006 Jan;74(1):88–98. doi: 10.1128/IAI.74.1.88-98.2006

FIG. 5.

FIG. 5.

Secretion analysis. In vitro expression and secretion of ESX-1 antigens from recombinant M. microti, BCG, and M. tuberculosis H37RvΔRD1 complemented with integrating cosmids that were mutated in selected RD1 genes was examined. Total protein concentrations were determined by using Bio-Rad protein assay, and 20-μg samples were subjected to SDS-PAGE. PPE68, CFP-10, and ESAT-6 correspond to products encoded by ppe68 (Rv3873), esxB (Rv3874), and esxA (Rv3875) genes, respectively. (A) CM, whole-cell extract containing cytosolic and membrane fractions; S, supernatant. Detection was carried out by using monoclonal anti-ESAT-6, polyclonal anti-CFP-10, and polyclonal anti-PPE68 antibodies. (B) Analysis of recombinant M. tuberculosis pe35 ko and control strains: (a) secretion analysis with same antibodies as described for panel A; (b) PCR control amplification obtained with primers for genes pe35 to Rv3877 for pe35 ko and RD1-2F9 M. tuberculosis H37RvΔRD1 strains; (c) hybridization signals obtained with radiolabeled cDNA from M. tuberculosis H37RvΔRD1::RD1-2F9 and pe35 ko mutant using a focused mini-array containing PCR products from selected genes of the RD1 region spotted on nylon membranes in duplicates. (C) Secretion analysis of recombinant M. tuberculosis strains lacking part of the ppe68 (Rv3873) gene or the described promoter region of esxB/A (rv3874-75) (4), with the same antibodies as described for panel A.