Inhibition of constitutive STAT6 phosphorylation in HL-derived cell lines by treatment with anti–IL-13.

(A) L-1236 cells were cultured for 18 hours in medium alone (lane 3), medium containing 20 μg/mL anti–IL-13 (lane 4), or medium containing 20 μg/mL isotype-matched control (lane 5). After 18 hours in medium alone, L-1236 cells were either left unstimulated for 15 minutes (lane 6), or treated for 15 minutes with 5 ng/mL IL-13 (lane 7) or 5 ng/mL IL-13 that had been preincubated for 1 hour with 20 μg/mL anti–IL-13 (lane 8). Equal amounts of total-cell lysates were then subjected to SDS–polyacrylamide gel electrophoresis, followed by Western blot analysis with antiphospho(Tyr641)–STAT6 (upper row). The membrane was stripped and reblotted with an antibody against total STAT6 to confirm equal loading (lower row). (B) HDLM-2 cells were cultured for 18 hours in medium alone (lane 3), medium containing 20 μg/mL anti–IL-13 (lane 4), or medium containing 20 μg/mL isotype-matched control (lane 5), and phospho-STAT6 levels were assessed as above. For both panels A and B, Daudi cells stimulated for 15 minutes with 10 ng/mL IL-4 served as the positive control (pos control, lane 1), and unstimulated Daudi cells served as the negative control (neg control, lane 2). These experiments were repeated twice with similar results.