Figure 2
Abnormal expression of EGFL7 mRNA and protein levels in FBs of SSc patients (A–C). FBs isolated from the skin of EOS SSc patients, express a statistically significant higher levels of EGFL7 mRNA, when compared to HC and LSS SSc-FBs (***p = 0.0001). The experiments were performed in triplicate for each patient and HC. Bars represent mean values ± SEM (N = 10 for each group) (A). 18 s gene served as the control. WB analysis of EGFL7 (B). These results confirm at protein level the results of qRT-PCR analysis. Blots were stripped using Re-Blot Plus Western Blot recycling kit (Chemicon International, USA) and re-probed with anti-mouse IgG β-actin antibody (Sigma-Aldrich, USA) to confirm similar loading of the gels and efficiency in electrophoretic transfer. The experiments were performed in triplicate for each patient and HC. Full length blot is represented in Supplementary Fig. 4. Immunoreactive bands were acquired by chemidoc (ImageLab). Densitometric analysis of the bands was performed using ImageJ software (NIH; online at http://rsbweb.nih.gov/ij) (C). IF on cultured FBs at 60% of confluence. Negative controls were obtained by omitting the primary antibody.
