Figure 3

Impaired ubiquitination of VEGFR2 and increased VEGF signaling in SSc-MSCs. (A) VEGFR2 was immunoprecipitated (IP) and its association with ubiquitin was assessed by western blot (WB). The immunoprecipitation assay showed that in HC-MSCs, after VEGF treatment, the VEGFR2 immunoprecipitated complexes contained ubiquitin. In SSc-MSCs a decreased level of Ubiquitin associated to the VEGFR2 was observed. (B) Proteins immunoprecipitated (IP) with VEGFR2 antibodies were fractionated by SDS-polyacrylamide gel electrophoresis. Immunoblots were probed with an antibody to phosphotyrosine (pY) and p85. After VEGF treatment, the tyrosine phosphorylation of VEGFR2 in SSc-MSCs was markedly increased when compared to HC-MSC. (C) Western blot of phosphorylated Akt and total Akt. In SSc-MSCs, after VEGF treatment, the levels of p-Akt were higher when compared to HC. Each experimental condition was performed in triplicate. The blots in A-B-C were representative of all the experiments. Protein bands were quantified by densitometry and the values were expressed as arbitrary unit of optical density (OD) (***P <0.0001).