Figure 4
SHP represses BMP-mediated SMAD1 transactivation via inhibition of its DNA binding.
(a,b) HepG2 cells were transfected with BRE-luc reporter plasmid along with vectors expressing SHP or ALK3 CA, and treated with BMP6 or metformin for 24 h. 3, 300 ng; 4, 400 ng; 6, 600 ng. (c) HepG2 cells were transfected with vectors expressing luciferase under the control of the hepcidin promoter, Hfe2 or SHP, and treated with metformin for 24 h. 3, 300 ng; 6, 600 ng. (d) Western blot analysis showing interaction of SHP and SMADs in 293T cells transfected with vectors expressing myc-SMAD1, myc-SMAD5, myc-SMAD8, HA-SMAD4 and Flag-SHP. The grouping of the image is from different parts of the same gel. (e) Q-PCR analysis showing hepcidin mRNA levels (top) and Western blot analysis showing SMAD1 expression (bottom) in mouse primary hepatocytes. Cells were transfected with siCon or siSMAD1 and infected with Ad-GFP or Ad-Flag-SHP. BMP6 was provided for 24 h+, 10 MOI. (f) Western blot analysis showing endogenous interaction of SHP and SMAD1 in HepG2 cells treated with BMP6 (20 nM) or metformin (2 mM) for 24 h. (g) Western blot analysis (top) and graphical representation (bottom) showing SHP effect on BMP6-medated SMAD1/5/8 phosphorylation. AML12 cells were infected with Ad-GFP or Ad-Flag-SHP (+10 MOI) and treated with BMP6 for 24 h. (h) ChIP assay was performed using soluble chromatin immunoprecipitated with anti-SMAD1/5/8, anti-SMAD4, and anti-Flag antibody. Mouse primary hepatocytes were treated with BMP6 for 24 h, and infected with Ad-GFP and Ad-Flag-SHP for 48 h. BMP-RE indicates BMP response element. Data are presented as means ± SD. Arrows show locations of molecular weight markers. The experiment was repeated on a minimum of three separate occasions. The western blot images were cropped with a grey cropping line. All gels for western blot analysis were run under the same experimental conditions. *P < 0.05, **P < 0.01 by two-tailed Student t-test.
