Hyb 76 8

The analysis of M. marinum protein secretion was carried out as previously described13 (link). For immunblot analysis, the membranes were stained with mouse monoclonal antibodies against the influenza hemagglutinin epitope (HA.11, Covance), anti-GroEL2 (CS44, John Belisle, NIH, Bethesda, MD, USA), anti-ESAT6 (Hyb 76-8; Statens Serum Institut, Copenhagen, Denmark) and mouse anti-PE_PGRS (7C4.1F7 7 (link)). The secondary horseradish peroxidase conjugated antibodies included goat anti-mouse IgGs (A106PS, American Qualex) or goat anti-rabbit IgDs (611-1302, Rockland), which were detected with chemiluminescence (Pierce).

Proteins were separated by SDS-PAGE (12.5% polyacrylamide) and transferred to a nitrocellulose membrane (GE Healthcare Life Sciences). Membranes were blocked for 1 h at RT. Proteins were labeled with primary mouse monoclonal antibodies anti-HA.11 (1:5,000), anti-EsxA (1:500, Hyb 76-8; Statens Serum Institut, Copenhagen, Denmark), anti-PE_PGRS (1:10,000,7C4.1F7) (5 (link)), anti-EccB₅ (1:5,000) (4 (link)), and anti-GroEL (1:10,000, CS44; John Belisle, NIH, Bethesda, MD, USA) for 1 h at RT. Secondary antibody goat anti-mouse IgG (1:2,500; Rockland) conjugated with horseradish peroxidase (HRP) was incubated for 1 h at RT, and proteins were visualized with ECL substrate (GE Healthcare Life Sciences). Protein concentrations were determined using the bicinchoninic acid assay (BCA assay; Thermo Scientific) for a proper normalization during loading.