Figure 4. The effect of the STAT3 ^M206K mutation on STAT3 translocation to the nucleus, transactivation activity, and cell proliferation.

(A) STAT3 phosphorylation (n = 3) was assessed in both nucleus and cytosol in stably transfected HEK-293T cells by mutant STAT3α^M206K and WT STAT3α plasmids. HDAC2 was used as a marker for nuclear protein, and GAPDH was used as a marker for cytoplasmic protein. (B) The transactivation activity of WT STAT3α^WT and mutant STAT3α^M206K was evaluated by luciferase reporter assay in Ly3 cells. Briefly, Ly3 cells were transiently transfected with plasmids STAT3α^WT and STAT3α^M206K along with a STAT3 luciferase reporter followed by IL-10 treatment for 6 hours and cells were used for luciferase assay. Bars represent mean ± SD from 3 replicates. Data was repeated three times (**P = 0.0042; ***P<0.001). The data were analyzed by the two-tailed unpaired Student's t test. (C) The effect of WT STAT3α^WT and mutant STAT3α^M206K on Ly3 cells proliferation by H³-thymidine incorporation assay. Bars represent mean ± SD from 4 replicates. The experiment was repeated three times (*P = 0.013). The data were analyzed by the two-tailed unpaired Student's t test.