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Figure 4 | BMC Biochemistry

Figure 4

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

Figure 4

Reciprocal aminoterminal interactions are not required for interdimeric protomer exchange. (A) Expression of tyrosine-phosphorylated GFP-tagged and untagged STAT1 and their corresponding F77A mutants in cellular extracts used for EMSA. A representative Western blot experiment using a STAT1-specific phospho-tyrosine antibody (top panel) and the corresponding re-blot after the stripping off of bound immunoreactivity and re-incubation with pan-STAT1 antibody C-24 (bottom panel) is shown. (B,C) Mutation of phenylalanine 77 to alanine does not interfere with the formation of heterodimeric STAT1 complexes. Extracts from U3A cells expressing wild-type or mutant STAT1 with and without the GFP-tag were co-incubated and the occupancy of the M67 element by heterodimers monitored over time using EMSA. A typical autoradiogram (B) and a quantification of three similar experiments (C) are shown. The histograms present means and standard deviations as well as significant differences over time. (D,E) Aminoterminal contacts between monomers are dispensable for the dissociation and re-association of STAT1 dimers. Extracts from U3A cells expressing either STAT1-GFP or untagged STAT1 were separately incubated in the presence of [33P]-2xGAS before being loaded together onto the gel (lanes 7 and 10) or incubated as a mixture in the presence (lanes 8 and 11) or absence of [33P]-2xGAS, which in the latter case was added immediately before gel electrophoresis (lanes 9 and 12). Reaction time was 45 min for all samples. An asterisk at the gel margin marks an unspecific band. (E) Quantification of band intensities corresponding to tetrameric STAT1 bound to tandem GAS sites across the indicated stoichiometry of GFP-tagged versus untagged STAT1 molecules. Numbers under each column give the ratio of STAT1-GFP/STAT1 molecules for each band. The protocol used for these experiments was similar to that shown in (D).

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