Western Blot Analysis of L. infantum

Western blot studies were performed as previously described (12 (link)). Briefly, total extract of L. infantum was obtained after growth of 1 × 10⁸ cells/ml until stationary phase. Cells were lysed using NP-40 lysis buffer supplemented with a protease inhibitor cocktail (2 mM AEBSF, 0.3 µM Aprotinina, 116 µM Bestatina, 14 µM E-64, 1 µM Leupeptina, and 1 mM EDTA; Sigma Adrich). After electrophoresis in a SDS-PAGE gel, samples were transferred to nitrocellulose membrane using the mini transblot electrophoretic transfer cell (Bio-Rad). Next, the membrane was blocked with 1% BSA prior to extensive washing in PBS Tween 0.1% (PBS-T). The blot was then incubated overnight with each respective primary antibody at a dilution of 1:250. Membranes were washed three times with PBS-T for 5 min and incubated with respective secondary antibodies conjugated to Alexa 488 (Santa Cruz Biotechnology). After washing three times with PBS-T for 5 min, the membranes were analyzed in the ChemiDoc MP (Bio-Rad).