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Figure 4 | Vascular Cell

Figure 4

From: The role of microRNAs in neural stem cell-supported endothelial morphogenesis

Figure 4

Altered expression of specific miRNAs in ECs effect expression of signaling proteins. A and B: Inhibition of miRNAs 155, 100, and let-7i in ECs induce the upregulation of components of the mTOR and TGF-β signaling pathways. ECs were transfected with synthetic inhibitors for miR-155, miR-100, and miR-let-7i, as well as Cy3 dye-labeled control anti-miR. At 24 hours post-transfection, EC lysates were subjected to immunoblot analysis using an mTOR signaling pathway antibody kit and antibodies against SMAD-2/SMAD-3, actin, and GAPDH. Protein expression was normalized by GAPDH expression and quantified using the GraphPad Prism software. A: Protein expression fold changes (anti-miR-transfected vs control anti-miR-transfected cells) are displayed graphically. B: Representative immunoblots for the data depicted on A. C and D: miRNA overexpression experiments. ECs were transfected with pre-miR miRNA negative control, miR-155, miR-100, and miR-let-7i-specific pre-miR miRNA precursors, or co-transfected with pre-miR-155 and pre-miR-100 (pre-miR-155+100). At 24 hours post-transfection, the cell lysates were immunoblotted with the antibodies against phospho-mTOR, IGF-1R (recognizing both pro- and mature form of the receptor), p53, and GAPDH (loading control). Protein expression was normalized by GAPDH expression and quantified using the GraphPad Prism software. C: Protein expression fold changes (pre-miR-transfected vs control pre-miR-transfected cells) are displayed graphically. Values represent the mean ± S.E.M. (n = 3, p < 0.05, Student's t-test). D: Representative immunoblots for the data depicted on C.

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