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Figure 3 | BMC Biochemistry

Figure 3

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

Figure 3

(A) Demonstration of STAT1 heterotetrameric complexes occupying a double-stranded oligonucleotide containing two consensus GAS sites. Cellular extracts from U3A cells expressing exclusively either GFP-tagged or untagged STAT1 were incubated separately with [33P]-labelled 2xGAS for 45 min (lanes 1 to 5) or, alternatively, mixed and co-incubated for 45 min (lane 6). Supershift reactions were performed by adding either an anti-STAT1 (lanes 2 and 4) or an unspecific anti-STAT3 antibody (lanes 1 and 3). Asterisks mark unspecific bands. (B) STAT1-GFP and STAT1 dimers compete for binding to 2xGAS. Different amounts of extracts from U3A cells expressing exclusively GFP-tagged or untagged STAT1 reacted either separately with [33P]-2xGAS or were mixed and reacted with the probe immediately before being loaded onto the gel. (C) Absence of binding to GAS elements promotes interdimeric protomer exchange. STAT1-GFP- and STAT1-containing extracts were co-incubated for 45 min in the presence (lane 1) or absence of [33P]-2xGAS (lane 2). Immediately before the reaction in lane 2 was loaded onto the gel, a similar amount of [33P]-2xGAS was added to the reaction as in lane 1. (D) Accumulation of interchanged STAT1 complexes at tandem GAS sites. Mixed STAT1-GFP- and STAT1-containing cellular extracts were incubated with [33P]-2xGAS for the indicated times, before being separated by gel electrophoresis (lanes 1 to 3). As controls, non-mixed extracts were used in lanes 4 and 5. (E) Time-dependent accumulation of tetrameric STAT1 at the expense of dimeric complexes. Histograms depict the ratio of tetrameric-to-total STAT1 binding activity presented as means and standard deviations from three independent experiments as shown in (D). Bars and asterisks indicate significant differences in the pattern of tetrameric-to-total GAS occupancy.

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