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Figure 4.
Figure 4. EMSA and footprinting of the binary and ternary
complexes in solution. (a) Titration of full-length NFAT RHR and
the NFAT RHR-N with the DNA used for crystallographic analysis.
Left panel, binding curve of full-length NFAT RHR binding to the
DNA fragment (K[d]=2.7(±0.4) nM, see Methods). Center
panel, EMSA gel of full-length NFAT RHR binding to the DNA
fragment. Right panel, EMSA gel of the NFAT RHR-N binding to the
DNA fragment. (b) Methylation interference footprinting of NFAT1
and NFAT1/Fos-Jun binding to ARRE2. F, free (unbound) DNA; L,
lower complex containing NFAT1 alone; U, upper complex
containing NFAT1 and Fos-Jun. Lanes 1-3, coding strand; 4-6,
non-coding strand. Fiducial lines connect certain residues to
their corresponding bands for clarity. (c) Ethyl nitroso-urea
(ENU) ethylation interference footprinting of NFAT1 and
NFAT1/Fos-Jun binding to ARRE2. F, free (unbound) DNA; L, lower
complex containing NFAT1 alone; U, upper complex containing
NFAT1 and Fos-Jun; G, (lanes 1 and 11) Maxam-Gilbert G lane;
lanes 2-4, coding strand using the full-length NFAT RHR; lanes
5-7, non-coding strand using only the NFAT RHR-N; lanes 8-10,
non-coding strand using the full-length NFAT RHR. Fiducial lines
connect certain residues to their corresponding bands for
clarity. On the right of the gel is a histogram showing
quantitative analysis of the footprints on the non-coding strand
for the NFAT1 RHR/DNA complex (open) and NFAT1 RHR/Fos-Jun/DNA
complex (shaded). Values are expressed as fractions of the
intensity at the same positions in the free probe after
normalization for loading.
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