Alteration of Differentiation Potentials by Modulating GATA Transcription Factors in Murine Embryonic Stem Cells
Figure 4
GATA-binding activity in retinoic acid-induced endoderm differentiation of wildtype and GATA-deficient ES cells. Wild type ES cells and those homozygous deficient in GATA4, GATA5, or GATA6 were treated with retinoic acid (1 M) for 4 days. Nuclear extracts of the ES cells were used for EMSA and supershifted with antibodies specific for each GATA factor. (a) For EMSA, the labeled Dab2-PI GATA binding probes were incubated with nuclear extracts from ES cells treated with retinoic acid (1 M) for 4 days. GATA6 (arrow) and GATA4 (arrowhead) containing complexes are indicated. The supershifted GATA4-containing complexes are indicated by a double-arrowhead. (b) GATA6 (−/−) ES cells in monolayers were transfected with GATA4 and immunofluorescence stainings of Dab2 (red) and GATA4 (green) were observed by fluorescence microscopy. DAPI (blue) was used for nuclear counter staining. (c) The binding to the PI probe is compared between nuclear extracts from retinoic acid-treated ES cells and cardiomyocytes. GATA4-containing (arrowhead), anti-GATA4 supershifted (double arrowhead), and GATA6-containing (arrow) complexes are indicated. Self competition (SP) assay was performed with nonradioactive PI probe and nonspecific competitions were performed with m1pI: PI probe with mutation in GATA binding site 1 (lane 6); with m2pI: PI probe with mutation in GATA binding site 2 (lane 7), and m1pI/m2 m2pI: probe PI with mutations in both GATA binding sites (lane 8). G4 and G6: antibodies against GATA4 and GATA6; SP: self competition with cold PI; NSP: nonspecific competition.
