Proteins from nuclear extracts from Hodgkin cell lines contain constitutively activated STAT3

. (A) Nuclear extracts (5 μg) of each cell line were analyzed in a gel retardation experiment using the radiolabeled STAT1/3 oligonucleotide. The DNA-protein complexes were separated by electrophoresis using a native 4.5% polyacrylamide gel as described in “Material and methods” and visualized by autoradiography. For comparison, nuclear extracts from unstimulated and IL-6–stimulated HepG2 cells were included in the study. Arrows show the position of STAT3 and STAT1 homodimers and heterodimers. (B) Western blot analysis of phosphorylated STAT3. Nuclear extracts (20 μg) of each cell line were analyzed in a 10% SDS-polyacrylamide gel, transferred to a nylon membrane, incubated with an antibody specific for phosphorylated STAT3 and visualized by ECL. (C) Western blot analysis of unphosphorylated STAT3. The same nylon membrane as in panel B was reprobed with an antibody specific for STAT3 and the protein was visualized by ECL.