Figure 5. Cell:cell contact but not conditioned media or IL-10 induce JAK2 phosphorylation.

Monocytes were either left untreated or treated with JSI-124 (as indicated above), and then were treated with either IL-10 (10 ng/ml), MCF-7's conditioned media or were co-cultured with MCF-7, for 2 hours. Untreated cells in the case of co-cultures were obtained by mixing MCF-7 and monocytes without incubation. Cell extracts were subjected to SDS-PAGE and anti-phosphorylated JAK2 immunoblotting (upper panels). Immunoblotting of anti-phosphorylated STAT3 demonstrates induction of STAT3 activation by all treatments regardless of JAK2 phosphorylation (middle panels) and that JSI-124 treatment selectively blocks contact- and JAK2-dependent STAT3 phosphorylation. Anti-STAT3 immunoblotting reveals relative amounts of protein in each lane (lower panels). Similar results were obtained in three independent experiments.