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. 2019 Jan 22;33(4):5599–5614. doi: 10.1096/fj.201802063RR

Figure 5.

Figure 5

HIP1 is a bona fide target of miR-135a-3p in ECs. A) Discovery and validation of miR-135a-3p target genes. HUVECs transfected with NSm and miR-135a-3pm were subjected to microarray gene profiling. Potential gene targets were further narrowed down by sequential use of bioinformatics and prediction algorithms, real-time qPCR, Western blot analyses, 3′-UTR reporter studies, and miRNP-IP analysis. B, C) HUVECs transfected with NSm or miR-135a-3pm were subjected to real-time qPCR for HIP1 expression (B) or Western blot analyses using antibodies to HIP1 and GAPDH (n = 3 experiments) (C). D) Luciferase activity of HIP1 3′-UTR normalized to total protein was quantified in HUVECs transfected with NSm or miR-135a-3pm (n = 3 experiments). E) MiRNP-IP analysis of enrichment of HIP1 mRNA in HUVECs transfected with NSm or miR-135a-3pm. RT-qPCR was performed to detect HIP1 or SMAD1. *P < 0.01. ns, nonsignificant. Results are representative of n = 3 replicates/group and 2 independent experiments. All data represent means ± sem.