The membranes were incubated with PbMLS and, subsequently, primary antibody anti-PbMLS and
secondary antibody anti-rabbit IgG. Negative control was obtained by incubating each protein extract with anti-PbMLS antibody, without preincubation with PbMLS (lanes 5, 6, 7 and 8). The numbers indicate the proteins (Additional file 2: Table S1) that interact with PbMLS that are confirmed by this technique. Another Far-Western Dibutyryl-cAMP nmr blot assay was performed using membranes that contained protein extracts of Paracoccidioides Pb01 mycelium, yeast, yeast secretions, and macrophage (Figure 2B, lanes 1, 2, 3 and 4, respectively). The membranes were incubated with PbMLS and, subsequently, were incubated with antibody anti-PbMLS and secondary antibody anti-rabbit IgG. Several proteins identified PX-478 purchase in the pull-down assays interacted with PbMLS at this point,
which suggested the veracity of the interactions. Negative control was obtained by incubating each protein extract with the anti-PbMLS antibody, without preincubation with PbMLS (Figure 2B, lanes 5, 6, 7 and 8). The numbers identify the proteins that interacted with PbMLS, as shown in Additional file 2: Table S1. PbMLS binds to the surface of macrophages Because the results from Far-Western blot assays revealed several macrophage proteins interacting with PbMLS, we performed immunofluorescence microscopy to visualize selleckchem whether PbMLS could adhere to the surface of the macrophage cells. No binding was observed using BSA as a control (Figure 3A). The arrow indicates PbMLS binding to a macrophage surface (Figure 3B). Figure 3 Binding of Pb MLS to the macrophage surface. Immunofluorescence microscopy that
shows the binding of PbMLS to J774 A.1 mouse macrophage cells. (A) Negative control was performed with the unrelated protein BSA. (B) Arrows indicate PbMLS (green) binding to the macrophage cell surfaces; blue indicates the macrophage nucleus. PbMLS participates in the adherence of Paracoccidioides to pneumocyte cells Because the fungus initially reaches the lungs, the participation of PbMLS in the adherence of Paracoccidioides Pb18 to pneumocyte cells was investigated by using confocal laser scanning microscopy. A549 cells were pretreated with anti-PbMLS Methocarbamol and infected with Paracoccidioides Pb18 isolate. After washings with frozen PBS-T, the monolayers were incubated with Alexa Fluor that was 594-conjugated for labeling the antibody. The arrows indicate PbMLS interacting with the A549 surface (Figures 4A and B). Figure 4 Interaction between Paracoccidioides yeast cells and pneumocytes by confocal laser scanning microscopy. Infected cell monolayers were fixed and permeabilized. Primary anti-PbMLS and secondary antibodies Alexa Fluor 594 goat anti-rabbit IgG (red) were used. The specimens were analyzed by laser confocal microscopy using DIC (A) and fluorescence (B).