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. 2016 Jan 11;291(11):5844–5859. doi: 10.1074/jbc.M115.675264

FIGURE 3.

FIGURE 3.

Sirt1 specifically regulates the PCs and CRH production and release in AtT20 cells. A–C, AtT20 cells were co-transfected with 4 μg each of preproPC2 cDNA and preproCRH cDNA alongside either GFP or Sirt1 cDNA and treated 48 h post-transfection with Ex-527 (50 μm) or vehicle control for 6 h. Western blot analysis was performed to measure Sirt1 and PC2 protein levels. A, Sirt1 protein (n = 8 GFP-vehicle (GV), 8 GFP-Ex527 (GE), 10 Sirt1-vehicle (SV), and 9 Sirt1-Ex527 (SE)). B, PC2 protein (n = 11 GFP-vehicle, 13 GFP-Ex527, 10 Sirt1-vehicle, and 10 Sirt1-Ex527). C, PC1 (n = 9 GFP-vehicle, 13 GFP-Ex527, 4 Sirt1-vehicle, and 9 Sirt1-Ex527). D–H, AtT20 cells were co-transfected with 4 μg of preproPC2 cDNA and preproCRH cDNA alongside either GFP or Sirt1 cDNA and protein lysates assessed for intracellular Sirt1 and PC2 protein and intracellular and released CRH content by Western blot (n = 6/group) and RIA, respectively. D, Sirt1 protein. E, PC2 protein. F, PC1 protein. These results were repeated in two independent experiments with the same response (n = 6/group). G, CRH content measured from protein lysates (n = 4 control and 6 Sirt1). H, released CRH content (n = 4 control and 6 Sirt1). Data are mean ± S.E. *, p value < 0.05; **, p value < 0.01; ***, p value < 0.001; ****, p value < 0.00001; #, p value < 0.05.