Phosphorylation of STAT proteins in HL and NHL cell lines.

Cell lysates were prepared from HL-derived cell lines (L-428, KM-H2, HDLM-2, L-1236, L-540), B-cell NHL-derived cell lines (Ly2, Ly10), a T-cell NHL-derived cell line (KARPAS-299), and B-cell lymphoblastoid cell lines (LCL-GK, LCL-HO). Equal amounts of whole-cell lysates were subjected to SDS–polyacrylamide gel electrophoresis followed by Western blot analysis. Phosphorylated STAT proteins were detected using phosphospecific antibodies recognizing Tyr641-phosphorylated STAT6, Tyr705-phosphorylated STAT3, and Tyr694-phsophorylated STAT5. These experiments were repeated twice with similar results. For P-STAT6, Daudi cells stimulated for 15 minutes with 10 ng/mL IL-4 served as the positive control, and unstimulated Daudi cells served as the negative control. For P-STAT3, serum-starved HeLa cells stimulated with IFN-α served as the positive control, and unstimulated serum-starved HeLa cells served as the negative control. For P-STAT5, TF-1 cells stimulated for 15 minutes with 25 ng/mL GM-CSF served as the positive control, and unstimulated TF-1 cells served as the negative control.