Detection of STAT3 Activation

To detect phosphor STAT3-Y705, total or nuclear protein extracts were analyzed by Western blotting using mouse monoclonal anti-Phospho-STAT3-Tyr705 antibodies (Cell Signaling) as described previously. To additionally measure STAT3 activity in cells, the Cignal STAT3 Reporter (luc) Assay (Qiagen) was applied. To this end, HepG2 or HepG2-H1.3 cells were transfected with either Cignal reporter or positive or negative control constructs using the fast-forward protocol provided by the manufacturer. The activity of STAT3-dependent Firefly luciferase and the activity of constitutively expressed Renilla luciferase used for internal normalization were measured by the Dual-Luciferase Reporter Assay System (Promega) according to manufacturer’s instructions.