Cotransfection of the IL-13Rα′ but not the IL-13Rα chain with the IL-4Rβ chain is sufficient to reconstitute STAT activation in response to IL-4. CHO-K1 cells were transfected with various chains and then incubated with IL-4 (50 ng/mL) for 10 minutes, washed with cold PBS, and solubilized with cold whole cell extraction buffer. Fifty micrograms of sample proteins were incubated for 20 minutes at room temperature with 1 ng of 32P-labeled SBE1 probe in binding buffer. Then, samples were loaded on a 4% nonreducing polyacrylamide gel and run at 150 V for 2 hours (A). For supershift assay, antimouse STAT6 (anti m-STAT6) or antihuman STAT6 (anti h-STAT6) rabbit polyclonal IgG was included in the reaction mixture before electrophoresis. The gel was dried and analyzed by autoradiography (B).
