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. 2015 Mar 12;112(13):3985–3990. doi: 10.1073/pnas.1503152112

Fig. 1.

Fig. 1.

STAT3 is dimethylated on K49. (A) STAT3-null A4 cells were infected with a retroviral construct expressing WT-STAT3, and stable pools of cells were selected with G418. The level of STAT3 expression, analyzed by the Western method, corresponds to that in the DLD1 parental cells. (B) A4-WT-STAT3 cells were treated with IL-6 (50 ng/mL) and soluble IL-6 receptor (sIL-6R) (62.5 ng/mL) for 1 h, and total cell lysates were analyzed for Y705 phosphorylation. (C) MS analysis of tryptic peptides of STAT3 indicates that K49 is dimethylated. Flag-tagged WT-STAT3 was immunoprecipitated from untreated or IL-6–treated A4-WT-STAT3 cells. The tryptic K49 peptide QFLAPWIESQDWAYAASK was included in the targeted analysis. A peptide consistent with a 28-Da mass modification of K49 was identified. (D) The relative abundance of the modified form of the K49 peptide was greater in the IL-6–treated samples. The levels of the modified forms of the peptides containing Y705, K49, and S727 were analyzed.