Fig. 4

Identification of miR-126 mRNA Targets

(A) Relative luciferase activity of constructs containing the 3′ UTR of potential miR-126 targets introduced into HeLa cells in the presence of miR-1 or miR-126. The 3′ UTR was also inserted in the antisense orientation as a control (control 3′ UTR). Firefly luciferase activity for each construct was normalized to the co-transfected Renilla luciferase construct and then normalized to the change in pGL3 luciferase in the presence of microRNA. For each 3′ UTR construct, normalized luciferase activity in the absence of microRNA was set to 1. ^* p < 0.05 compared to pGL3.

(B) Relative luciferase activity of select constructs in (A) in HUVECs upon inhibition of miR-126 with MOs. Normalization was performed as in (A).

(C) mRNA levels of potential targets in HUVECs transfected with miR-126 mimic or MO quantified by qRT-PCR. Values are relative to transfection controls.

(D) Immunoblot of SPRED1, VCAM1, and PIK3R2 protein in control or miR-126 MO-transfected HUVECs 72 hr post-transfection. GAPDH is shown as a loading control.

(E) Sequence complementarity of a potential miR-126 binding site in the zebrafish spred1 3′ UTR.

(F and G) Luciferase assays (as in (A) and (B), respectively) using the zebrafish spred1 3′ UTR.