Immunoblotting Analysis of STAT Signaling

Cell lysis, protein isolation and immunoblotting were performed as described previously [24 (link)]. Twenty micrograms of protein were resolved using 4–12% Bis-Tris polyacrylamide gels. Gels were transferred to PVDF membranes and subjected to blocking and incubation with primary and secondary antibodies. The primary antibodies used in this study included: phospho-STAT3 (Tyr705, Cell Signaling, Boston, MA and Abcam, Cambridge, MA), total STAT3 (Cell Signaling and Abcam), phospho-STAT1 (Cell Signaling), total STAT1 (Cell Signaling), phospho-STAT2 (Cell Signaling), phospho-STAT4 (Cell Signaling), phospho-STAT6 (Cell Signaling), and GAPDH (Cell Signaling). Protein bands were detected using Western Lightning ® Plus Enhanced Chemiluminescence substrate (Perkin Elmer, Waltham, MA) and HyBlot CL ® Autoradiography film (Denville Scientific, Holliston, MA). The cytokines used in this study included: interferon gamma (IFN-γ, Cell Signaling), interferon alpha (IFN-α, Cell Signaling), interleukin-4 (IL-4, Cell Signaling), interleukin-6 (IL-6, Cell Signaling), and oncostatin M (OSM, Cell Signaling).