A: Testosterone administration for 48h upregulated the expression of androgen receptor (AR) and phospho-Smad1, but had no effect on the expression of total Smad1 and Smad4. The results are representative of 3 experiments. Each lane represents the result from one animal.
B: Liver nuclear extracts were immunoprecipitated with goat-anti-Smad4 (left panel) or rabbit-anti-AR (right panel) antibodies. Immune complexes were separated by gel electrophoresis and detected using anti-Smad1, anti-Smad4, anti-CBP/p300, and anti-AR antibodies. Immune complexes immunoprecipitated with anti-Smad4 antibody contained AR, Smad1 and p300 proteins (left panel). Similarly, Immune complexes immunoprecipitated with anti-AR antibody contained Smad4, Smad1, and p300 (right panel). All samples were pre-cleared with agarose gel conjugated with normal goat IgG (for ip with Smad4) or rabbit IgG (for ip with AR) before first antibody was added to the reaction. Equal inputs were confirmed by Western blot for LaminA/C (not shown). Results are representative of 4 experiments. These data provide evidence of association of AR with Smad1, Smad4, and CBP/p300.
C: Liver hepcidin mRNA expression in mice treated with vehicle (C), dorsomorphin (DM) (an inhibitor of BMP/Smad1 signaling), DM plus testosterone (DM+T), or T alone. Administration of DM and T each down-regulated liver hepcidin mRNA expression. However, combination of both did not decrease it any more than T alone, suggesting that T and DM likely share overlapping pathways for hepcidin regulation. Results are mean± SEM, N=4 for each group.
D: Liver tissue isolated from mice treated with vehicle (C) or testosterone (T) was subjected to ChIP analysis. Immuno-precipitation of Smad1 protein-DNA complexes was performed using anti-Smad1 (Cell Signaling#6944). Negative controls (Neg) were immuno-precipitated with rabbit IgG and positive controls (Pos) were immuno-precipitated with rabbit anti-histone 3 (H3). Real-time PCR was performed using primer sets flanking BMP-RE1 and BMP-RE2 of mouse hepcidin promoter. Results were normalized to the corresponding inputs (sonicated chromatin before immuno-precipitation). Treatment with testosterone for 48h reduced the association between Smad1 and the BMP-RE1 (left panel) and BMP-RE2 (middle panel) on the hepcidin promoter. The assay specificity was validated by primers designed for RPL3 intron 2 (Cell Signaling, #7015) which strongly binds to H3 but not Smad1 or rabbit IgG (right panel). Results are mean±SEM, N=3 for each group.