Proteins that presented the major phosphorylation changes in response to TGF-β.

<p>Magnified regions showing protein spots that presented the greatest up- (A) or down-(C) phosphoregulation and their respective bar graphics (B and D) with the mean fold change values.</p

Kinetic view of protein expression in response to TGF-β.

<p>Expression pattern of elongation factor 1-α (A), β-tubulin (B) and cruzipain (C) from <i>T. cruzi</i> epimastigotes incubated with TGF-β at different periods of time. The graphics represent the values found for the kinetic of these proteins obtained through a 2DE approach and Western blot analysis.</p

Distribution of the identified proteins into functional groups.

<p>Bar graphs presenting the functional groups of proteins that had their expression (red) or phosphorylation (green) regulated by TGF-β.</p

Biological effects exerted by TGF-β over different forms of <i>T. cruzi</i>.

<p>A. <i>In vitro</i> proliferation of epimastigotes. Epimastigotes were grown in the absence or presence of TGF-β (5 ng/ml, daily added) in LIT medium. The graph represents the mean number of epimastigotes present in cultures treated or not with TGF-β after 24, 48, 72 and 96 hours. N=4. **p<0.01, ***p<0.001. B. Amastigogenesis <i>in vitro</i>. Differentiation of trypomastigotes was performed by acid induction followed or not by treatment with 5 ng/ml of recombinant TGF-β 1. The graph represents the percentage of differentiation into amastigote forms after 4 hours of acid induction with or without TGF-β stimuli. N=4. **p<0.01.</p

Proteomic map of <i>T. cruzi</i> epimastigotes.

<p>The image shows the total protein pattern of epimastigotes treated with TGF-β for 1 minute and separated by 2D-PAGE (17 cm IPG strips in the pH range 3-10NL and 12% SDS-PAGE, CBB-G250 staining). The colored circles indicate protein spots identified by mass spectrometry that differ significantly only in their phosphorylation (green), or in their expression (red) or in both (brown) levels in one or more studied time points. The molecular weight (MW) of marker proteins and the pH range of the IEF gradient are indicated. N=3.</p

TGF-β responsive proteins identified by MALDI TOF-TOF.

<p>pI theor = theoretical isoelectric point.</p><p>pI exp = experimental isoelectric point.</p><p>MW theor = theoretical molecular weight.</p><p>MW exp = experimental molecular weight.</p

TGF-β induces cruzipain expression in <i>T. cruzi</i> epimastigotes.

<p>Parasites treated (B) or not with TGF-β (A) for 5 minutes were immunostained for cruzipain and corresponding histograms were obtained using the ImageJ software (C and D). The quantification of the parasite areas labeled for cruzipain were determinate by the mean of RGB color and demonstrated as a bar graphic (E).</p

Bidimensional phosphoproteome of <i>T. cruzi</i> Y strain epimastigotes.

<p>Representative images show the total protein patterns of <i>T. cruzi</i> treated or not with TGF-β for 1, 5, 30 or 60 minutes. Protein extracts (100 µg) were separated on 7 cm pH 3–10NL IPG strips and 12% SDS-PAGE gels. Phosphoproteins were stained with Pro-Q Diamond (shown in green) and total protein stained with Sypro Ruby (shown in red). Images were artificially colored using PD Quest (BioRad) software tools. Phosphoprotein staining was confirmed with the visualization of the two positive molecular weight control bands (Peppermint Stick – Molecular Probes). The molecular weight (MW) of marker proteins and the pH range of the IEF gradient are indicated. N=3.</p

Proteins with changes in phosphorylation pattern in response to TGF-β.

<p>Fold changes presents the ratio compared with values in the absence of TGF-β stimulation.</p>*<p>Values equal to zero correspond to spots that could not be detected on gels.</p

Proteins that presented the major expression changes in response to TGF-β.

<p>Magnified regions showing protein spots that presented the greatest up- (A) or down-(C) regulation in expression and their respective bar graphics (B and D) with the mean fold change values.</p