Preparation of cell lysates, supernatants, and western blotting.
Protein preparation of whole cell lysates and culture supernatants of mycobacterial cultures was done as previously described [24]. Total protein concentrations were determined by using a Bio-Rad protein assay (Bio-Rad), and 15-μg samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using NuPage Bis-Tris Mini gels (Invitrogen) in NuPage MES running buffer following the manufacturer's recommendations. The immunoblot analysis was carried out with mouse ESAT-6 monoclonal antibody (Hyb 76–8; Antibody Shop, Statens Serum Institute) as described previously [24], after proteins were transferred onto nitrocellulose membranes using the iBlot Dry Blotting System (Invitrogen; Voltage 25V; Run time: 7 min).