Figure 8.

Evaluation of blocking peptide capacity to inhibit integrin α4β1 signaling and downstream profibrotic effects. NIH/3T3 fibroblasts were incubated in the presence or absence of 10 ng/mL TGF-β1 with or without 10 µg/mL AF38Pep for (A) 1 h or (B) 12 h, before assessment of extracellular-signal-regulated kinases 1 and 2 (ERK1/2) and focal adhesion kinase (FAK) protein expression and phosphorylation. Immunoblots are displayed alongside corresponding densitometry analysis of phospho-protein/total-protein and total-protein/GAPDH loading control. (C) Gelatinase zymography indicating pro-matrix metalloproteinase 9 (pro-MMP9) (top band) and pro-MMP2 (bottom band) enzymatic activity following the indicated treatments for 48 h. Shown alongside is the quantification of relative gelatinase activity. (D) The mRNA expression for genes associated with integrin α₄β₁ activation: Mmp9, Tgfb1, Ltbp1, Hic5, and Mrtfa, which were assessed by real-time quantitative PCR (qRT-PCR) following 48 h of stimulation by 10 ng/mL TGF-β1. Blots, images, and data are representative of three independent experiments and data are shown as the mean ± S.E. Statistical analysis is shown as *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05, and ns = no statistical significance (p > 0.05).