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. 2019 Nov 1;33(12):14636–14652. doi: 10.1096/fj.201901930R

Figure 3.

Figure 3

HuR and AUF1 bind directly to the 3′–UTR of Nrf2-mRNA in cells. A) RIP was carried out by expressing flag-tagged HuR or AUF1 in HEK293T cells and subjecting lysates to Flag IP. B) Nrf2-mRNA coeluting with enriched proteins was detected with real-time qPCR (mean ± sem of n = 4 independent replicates). C) 3′–UTR reporter system consisting of Nrf2-mRNA fused to a firefly luciferase transcript and normalized to Renilla luciferase. D, E) Knockdown of HuR and AUF1, respectively, reduces the 3′–UTR reporter activity in HEK293T cells (mean ± sem of n = 8 independent replicates for each bar). F) Knockdown of both proteins leads to a further suppression of the 3′–UTR reporter activity (mean ± sem of n = 4 independent replicates). See also Supplemental Fig. S7B. All P values were calculated with Student’s t test. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; ZNF200, zinc finger protein 200.