A 996-bp segment of the human EGR1 3′UTR containing the miR-146a site was cloned into the pmiRGlo dual luciferase vector (Promega). A similar cloning strategy was used to clone murine Egr1 3′UTR and the Nrp2 UTR (see Supplementary Table 1 ). For mutation of the miR-146a binding site, we utilized site-directed mutagenesis as previously described using the primers shown in Supplementary Table 1 [43 (link)]. Co-transfections were performed with Lipofectamine 2000 (Life Technologies) as per the manufacturer's instructions. Cells were lysed after 24 hours, substrate was added and luminescence was measured on a Glomax-Multi Jr (Promega).
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