Kruppel like factor expression and DNA binding activity during ES cell differentiation. (A) Co-expression of Oct4 with KLF2 or KLF4 in ES cells. Indirect immuno-fluorescence shows co-localization of KLF2 and KLF4 with Oct4 in sub-nuclear compartments (possibly nucleoli). Individual confocal images for OCT4, KLF2, KLF4, and DAPI are shown with the corresponding composite image. Scale bar 40 μm. (B) Electro-mobility gel shift assay showing changes in DNA binding activities at a conserved CACC box site in the p18-INK4c gene promoter. Nuclear extracts were generated from ES cells or EBs differentiated for four days in serum. Super-shifts were performed with specific antisera for SP1, SP3, KLF2, KLF3, and KLF4 (See Methods). There is strong binding of endogenous Sp1 to the CACC element in ES cells and EB cells. KLF2 DNA-binding activity is present in ES cells as determined by a specific inhibition of binding of the indicated DNA complex with a KLF2 antibody. This activity is lost upon differentiation into EBs. The identity of the CACC box binding activity in EBs denoted CAC-X, and the binding activity in ES cells denoted CAC-Y, was not definitively identified using this panel of antibodies.