FIG. 4.

Interaction of between SHP and STAT5 in vivo. A: Wild-type mice were injected with GH (2 μg/g body weight) for 1 h or metformin (Met; 200 mg/kg body weight) for 6 h at the indicated concentrations. Coimmunoprecipitation assays with liver extracts demonstrate the functional association between SHP and STAT5. Tissue extracts (700 μg/lane) were immunoprecipitated with STAT5 antibody and blotted with SHP antibody. Expression of p-STAT5, STAT5, and SHP from 10% input were analyzed by immunoblotting. All mice were separated into experimental groups (n = 4–6 mice/group). B: In vivo interaction between SHP and STAT5. HepG2 cells were cotransfected with expression vectors for STAT5 together with p-EBG-SHP (GST-SHP) and p-EBG (GST alone) as a control. The complex formation (top) and the amount of STAT5 used for the in vivo binding assay (bottom, lysate) were analyzed by Western blot (WB) using anti-STAT5 antibody. The same blot was stripped and reprobed with an anti-GST antibody (middle) to confirm the expression levels of the GST-SHP and the GST control. C: Schematic diagrams show the wild-type (wt) and the deletion forms of the mPDK4 promoter constructs. HepG2 cells were cotransfected with wild-type, deletion form mPDK4 reporter, and TEL-JAK2, STAT5, respectively. After transfection for 36 h, cells were treated with GH (500 ng/mL) for 1 h. Luciferase (Luc) activity was normalized to β-galactosidase activity to correct for transfection efficiency. All data are shown as fold activations relative the control (± SEM). D: ChIP assay. Rat primary hepatocytes were infected with Ad-siSHP for 36 h and then treated with GH and metformin at the indicated concentrations. Before immunoprecipitation, an aliquot of the sample was stored and then purified and represents input for each sample. Cell extracts were immunoprecipitated with STAT5 and SHP antibodies, and purified DNA samples were used to perform PCR using primers binding the specific proximal (left) and nonspecific distal (right) regions on the mPDK4 gene promoter. All data are representative of at least three independent experiments.