Fig. 2

a p-STAT3 phospho-specific immunoblot of four ES cell lines. rhIL6-mediated STAT3 activation was detected after 20 min incubation of indicated IL6 concentrations in cell lines A4573, TC32 and CHP. All cell lines showed low intrinsic phosphorylation of STAT3. Cell line 5838 did not display an increase in p-STAT3 after incubation with rhIL6. b p-STAT3 phospho-specific immunoblot of cell line A4573 after incubation with 50 ng/ml rhIL6 for indicated time periods. Maximal levels of phosphorylated STAT3 were seen 20 min after adding rhIL6. STAT3 phosphorylation decreased after 2 h of incubation. c Phosphorylation of STAT3 in A4573 ES cell line is IL6-specific. Cells were stimulated with rhIL6 (100 ng/ml for 20 min). An IL6 receptor-specific antagonistic antibody (2 μg/ml) was added 30 min prior to rhIL6 incubation where indicated. The prostate cancer cell line LNCaP was used as a positive control. STAT3 phosphorylation mediated by rhIL6 could be blocked by an anti-IL6 receptor antibody. Data representative for three independent protein harvests and immunoblots