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. 2016 Nov 17;7(11):e2473. doi: 10.1038/cddis.2016.341

Figure 4.

Figure 4

Western blot, CHIP assay and siRNA silencing demonstrated that Nrf-1 and P-CREB1 cooperate to promote transcription of ligase IV in retinal neurocytes. (a) Retinal expression pattern of Nrf-1, P-CREB1 and ligase IV in retinal neurocytes in vitro. Western blotting indicates that lithium (1.0 mM) alters the expression level of Nrf-1, P-CREB1 and ligase IV in retinal neurocytes only under conditions of serum starvation. (b) Sonicated DNA isolated from retinal neurocytes treated with formaldehyde to crosslink endogenous proteins to DNA (fragments averaged ~0.5 kb in length). (c) CHIP assayed for P-CREB1, Nrf-1 antibody and a control normal rabbit IgG, and PCR amplified a 360- or 305-bp fragment of ligase IV promoter spanning the ATF sites. The amplified PCR fragments were analyzed on a 2% agarose gel. (d) siRNA silencing markedly decreased CREB1 and Nrf-1 expression in retinal neurocytes. In accordance, the siRNA abolished the lithium-stimulated increase in ligase IV expression. (e) Silencing of CREB1 and Nrf-1 significantly inhibited the increase in promoter activity in lithium-treated cells (Con, 1; Li, 2.2±0.47; Li+siRNA, 1.27±0.03). n=3 for each group, *P<0.05, **P<0.001