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Figure 2 | BMC Biochemistry

Figure 2

From: DNA binding reduces the dissociation rate of STAT1 dimers and impairs the interdimeric exchange of protomers

Figure 2

(A) Electrophoretic mobility shift assay (EMSA) for the identification of green-fluorescent protein-tagged STAT1 (STAT1-GFP) and untagged STAT1. Extracts from reconstituted STAT1-negative U3A cells expressing recombinant GFP-tagged or untagged STAT1 were incubated at room temperature with a [33P]-labelled double-stranded oligonucleotide containing two GAS sites in tandem orientation (2xGAS). Supershift reactions were performed by adding anti-STAT1 antibody C-24 (lanes 1 and 3), and, as control, an unspecific STAT3 antibody (lanes 2 and 4). For competition, a 750-fold molar excess of unlabelled GAS was added to the reaction (lane 5). Asterisks mark unspecific bands. (B,C) Similar dissociation kinetics of STAT1-GFP and STAT1 from a single consensus GAS site (M67). Cellular extracts from U3A cells expressing tagged or untagged STAT1 were incubated for 15 min with [33P]-labelled M67 before on ice a 750-fold molar excess of unlabelled M67 was added for 0, 5, and 10 min, respectively. (B) Specific DNA-binding activity was detected by autoradiography in vacuum-dried gels. (C) The histograms demonstrate the decline in specific DNA-binding activity during challenge with unlabelled M67 for STAT1-GFP (black columns) and untagged STAT1 (grey columns). Bars and asterisks indicate significant differences between samples over time.

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