*2.3. Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis and Western Blot Analysis*

Cell lysates of mid-logarithmic phase BCG transformants were prepared by sonication in a protein extraction buffer (50 mmol/L Tris–HCl pH 7.5, 5 mmol/L Ethylenediaminetetraacetic acid (EDTA), 0.6% sodium dodecyl sulfate) containing protease inhibitor cocktail (Sigma-Aldrich, St Louis, MO, USA). Cell lysates supernatants were subsequently separated by a Novex 4–12% Bis-Tris SDS-PAGE gel (Thermo Fisher Scientific, Waltham, MA, USA), and electroblotted onto a pretreated polyvinylidene difluoride membrane using an iBlot kit (Thermo Fisher Scientific, Waltham, MA, USA). The HTI protein was stained using the primary anti-HTI monoclonal antibodies n63 and n69 at 5 μg/mL overnight kindly provided by Aelix Therapeutics (Barcelona) followed by secondary goat anti-mouse Immunoglobulin G-Horse Radish Peroxidase(IgG–HRP) antibody (Jackson ImmunoResearch, Cambridgeshire, UK) for 1 h diluted at 1:10,000. The membrane was developed using the SuperSignal™ West Femto Maximum Sensitivity Substrate kit (Thermo Fisher Scientific, Waltham, MA, USA).
