Ba/F3–IL-6R (106) and Ba/F3-gp130 cells/ml and variants thereof were washed and starved in serum-free medium for 6 hours. Afterward, cells were stimulated with the indicated cytokines or inhibitors for 30 min [IL-3 (10 ng/ml), HIL-6 (10 ng/ml), sgp130Fc (10 μg/ml), tocilizumab (10 μg/ml), B-R3 (1 μg/ml), P6 (10 mM), and GW (3 μM)]. The cells were harvested by centrifugation at 4°C for 5 min at 1500g. Cells were suspended in Laemmli sample buffer, and pSTAT3 analysis was done by Western blotting as described.

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