Effect of JAK2 inhibition on JAK/STAT signaling. (A) DHL2 cells were treated with various doses of the JAK2 inhibitor TG for 24 hours and phosphorylation of JAK1, JAK2, and STAT3 was analyzed. (B) Ly3 and DHL2 cells were treated with a 4μM concentration of TG for 2, 4, and 8 hours and phosphorylation of STAT3 was performed by Western blotting. (C) Ly3 and DHL2 cells were treated with neutralizing IL-10 Ab for 24 hours and STAT3 phosphorylation was detected. (D-F) DHL2 cells were exposed to various concentrations of TG for 8 hours and then lysed. Cell lysates were then immunoprecipitated with anti-JAK1, anti-JAK2, or anti-STAT3 and blots were probed with phosphotyrosine Ab. DHL2 (G) and DHL2 and LY3 (H) cells were pretreated with 100 ng/mL of rIL-10 for 30 minutes and then treated with various concentrations of TG for 4 hours (G) and 24 hours (H), and JAK2 and STAT3 phosphorylation was analyzed by Western blotting.