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. 2015 Feb 17;112(9):2699–2704. doi: 10.1073/pnas.1424641112

Fig. 2.

Fig. 2.

Ethanol disrupts recruitment of CREB and CRTC2 to gluconeogenic promoters. (A) Effect of a 10-min pretreatment with ethanol (EtOH) on gluconeogenic gene expression in mouse primary hepatocytes exposed to glucagon for 1.5 h. Each bar represents averaged results for three biological replicates, assayed three times each. Error bars indicate SEM. *P < 0.05; **P < 0.01; ***P < 0.001. (B) Western blot showing the effect of a 10-min pretreatment with ethanol on phosphorylation of CREB and LKB and dephosphorylation of CRTC2 in primary hepatocytes exposed to glucagon for 45 min. CREB and HSP90 served as loading controls. (C) Immunofluorescence staining showing the effect of a 10-min pretreatment with ethanol (250 mM) on the nuclear translocation of CRTC2 in primary hepatocytes exposed to glucagon for 45 min. Red, CRTC2; green, actin; blue, DAPI. (D) Effect of a 10-min pretreatment with ethanol on G6pc-Luc and CRE-Luc reporter activities in primary hepatocytes exposed to glucagon for 5 h. Each bar represents averaged results for three biological replicates, assayed three times each. Error bars indicate SEM. *P < 0.05; **P < 0.01; ***P < 0.001. (E) ChIP assays showing the effects of a 10-min pretreatment with ethanol (250 mM) on the recruitment of CREB, phospho-CREB, and CRTC2 to CREB-binding sites over gluconeogenic (G6pc, Pck1) promoters in primary hepatocytes exposed to glucagon for 1 h. The 36b4 ribosomal protein gene served as a negative control. Each bar represents averaged results for two biological replicates, assayed three times each. Error bars indicate SEM. *P < 0.05; **P < 0.01; ***P < 0.001.