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. 2018 Jul 13;293(36):14001–14011. doi: 10.1074/jbc.RA118.003099

Figure 4.

Figure 4.

Nur77 is involved in the ISO-induced RLN3 expression in cardiomyocytes. A, NRVMs cultured in 12-well plates were transiently transfected with either 200 ng of RLN3 promoter or 400 ng of mutant luciferase reporter vector, 20 ng of pCMV-R.Luc. 48 h after transfection, cells were stimulated with the indicated concentration of ISO for 6 h. Cells were washed once with ice-cold PBS and then analyzed for luciferase activities using the Dual-Luciferase reporter assay system (Promega) (n = 6). *, p < 0.05 compared with ISO at 0 μm. B, NRVMs were stimulated with 10 μm ISO for 3 h, and the nuclear extracts (NE) were then isolated and used for EMSA to detect the interaction of Nur77 with the NBRE in the RLN3 promoter. A 100-fold excess of unlabeled competitor and anti-Nur77 antibody was used to demonstrate the specificity of the shifted complex. C, myocytes were stimulated with 10 μm ISO for 3 h, and the recruitment of Nur77 to the RLN3 promoter was determined by ChIP assays using anti-Nur77 antibody. *, p < 0.05 compared with ISO at 0 μm. Error bars, S.D.