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. 2010 Nov 23;107(50):21499–21504. doi: 10.1073/pnas.1016147107

Fig. 2.

Fig. 2.

Nuclear import and DNA binding are required for IL-6–induced S727 phosphorylation and K140 dimethylation of STAT3. (A) Western analyses for STAT3, S727-phosphoryl-STAT3, and STAT3-K140me2. STAT3-null A4 cells were infected with retroviral constructs and stable pools were selected with G418. A4, STAT3-null cells; WT, YF, S727A, K140A, K140R, R214/215A and R414/417A, A4 cells expressing normal levels of wild-type or mutant STAT3 proteins. Total lysates prepared from cells treated with IL-6 for 4 h were analyzed by Western blot. (B) A4 cells expressing wild-type STAT3 were treated with IL-6. Cytoplasmic and nuclear fractions representing equal numbers of cells were separated by electrophoresis and analyzed by Western blot. (C) A4 cells expressing wild-type STAT3 were grown on coverslips to 20 to 30% confluence, then treated with IL-6 for 4 h, followed by staining with primary antibodies directed against STAT3, S727-phosphoryl-STAT3 and STAT3-K140me2. Following staining with DAPI (blue nuclear stain) and fluorescent secondary antibodies against STAT3 (green), S727-phosphoryl-STAT3 (red) or STAT3-K140me2 (red), the cells were examined by confocal microscopy. The yellow/pink pixels in the composite image demonstrate the close association of the two proteins. (D) Northern analysis of gene expression. Cells were treated with IL-6 as above for 4 h and total RNAs were analyzed for GAPDH and SOCS3.