Immunoblotting was performed using the anti-Pb14-3-3 polyclonal antibody to verify 14-3-3 protein reactivity.

<p>Cytoplasmic fraction from <i>P. brasiliensis</i> grown in Fava Nettós media (1), cell wall fraction from <i>P. brasiliensis</i> grown in Fava Nettós media (2), and A549 cells infected with <i>P. brasiliensis</i> for 2 h (4), 5 h (6) and 8 h (8). The control was performed using noninfected A549 cells for 2 h (3), 5 h (5), and 8 h (7).</p

Inhibition of the interaction of <b><i>P.</i>brasiliensis</b> with epithelial cells using polyclonal anti-14-3-3 produced in rabbits at different times.

<p>*p≤0.05 compared to untreated cells with cells infected with <i>P. brasiliensis</i> previously treated with polyclonal anti-14-3-3.</p

Comparison of the 30 kDa adhesin with the 14-3-3 protein of <i>P.brasiliensis</i> by BLASTp and FASTA 3.

<p>Comparison of the 30 kDa adhesin with the 14-3-3 protein of <i>P.brasiliensis</i> by BLASTp and FASTA 3.</p

IEM detection of the 14-3-3 protein of <i>P.brasiliensis</i> during interaction with epithelial cells (A549) for 2 h (A), 5 h (B) and 8 h (C).

<p>Magnification 50,000×. F: fungus, W: fungal cell wall, V: vacuole, C: epithelial cell.</p

Immunoelectron microscopy to determine the localization of the 14-3-3 protein in yeast cells of <i>P. brasiliensis</i>.

<p>(A) The negative control was performed with pre-immune serum. (B) and (C) Labeling with the polyclonal anti-14-3-3 antibody. The arrows indicate the gold particles, demonstrating the sub-cellular localization of this protein. Magnification 25,000×.</p

Inhibition assay of the interaction between <i>P.brasiliensis</i> and epithelial cells using recombinant 14-3-3 protein at different times.

<p>*p≤0.05 compared to untreated cells.</p

SDS-PAGE to verify protein induction (A) and 14-3-3 recombinant protein purification (B).

<p>Gels were stained with Phast Gel Coomassie R350. (A) 1: LMW ladder (low molecular weight, GE Life Science), 2∶5 h of induction with 0.4 mM IPTG at 37°C. (B) 1: LMW, 2: purified protein. The arrow indicates the purified 14-3-3 recombinant protein. (C) Immunoblot to verify the reactivity of polyclonal serum anti-14-3-3. (1) Pre-immune serum 1∶100– control, (2) full recombinant 14-3-3 protein and (3) cleaved 14-3-3 protein induced for 2 h.</p