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. 2007 Oct 15;27(24):8547–8560. doi: 10.1128/MCB.00589-07

FIG. 1.

FIG. 1.

Sumoylation of EKLF in vivo. (A) Schematic representation of EKLF domains with posttranslational modifications and potential sumoylation site. Below is the alignment of amino acid sequences of EKLF from humans, chimps, mice, and rats spanning the SUMO consensus motif. (B) Mouse EKLF is sumoylated on lysine 74. 293T cells were cotransfected with constructs encoding Flag-EKLF (WT or K74R), Ubc9, Flag-PIAS1, and HA- or GFP-tagged SUMO. Lysates were subjected to immunoprecipitation (IP) with M2-agarose, and precipitated proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blotted (WB), and probed with anti-EKLF antibody (Ab). (C) Sumoylation of endogenous EKLF in erythroid cells. MEL cells were stably transfected with plasmids encoding Flag-WT EKLF or Flag-K74R EKLF. IP was carried out as for panel B, and the blots were probed with anti-EKLF or anti-SUMO antibodies as indicated. (D) Sumoylation of endogenous EKLF from fetal liver at E13.5. Protein extracts form fetal liver were subjected to IP with monoclonal EKLF antibody 7B2, and precipitated proteins were separated by SDS-PAGE, blotted, and probed with either polyclonal anti-EKLF or anti-SUMO antibodies.