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. 2015 Sep 2;89(22):11487–11499. doi: 10.1128/JVI.01887-15

FIG 6.

FIG 6

Affinity analysis. (A) Analytical-exclusion chromatograms of ECFP-fused STAT1ND (gray line) and EYFP-fused Y3 (black line). The broken line shows a 1:1 mixture of ECFP-fused STAT1ND and EYFP-fused Y3. A 15 μM concentration of ECFP-fused STAT1ND was preincubated with or without an equimolar concentration of EYFP-fused Y3 for 10 min, followed by gel filtration chromatography using a Superdex 75 10/300 GL high-performance liquid chromatography (HPLC) column. (B and C) Fluorescence emission spectra by direct titration to 0.2 μM ECFP-fused STAT1ND (B) or 0.05 μM unfused ECFP (C) with EYFP-fused Y3 at 0.16, 0.32, 0.48, 0.64, 0.80, 0.96, and 1.12 μM (gray line). Two arrows represent decreased fluorescence intensity at 475 nm and increased fluorescence intensity at 527 nm. rfu, relative fluorescence units. (D and E) The quenched fluorescence intensities (ΔI) of ECFP-fused STAT1ND (D) or STAT1(1–713) (E) at 475 nm caused by the addition of EYFP-fused Y3 are divided by the concentration of each STAT1 dimer. The values are then plotted against the concentration of free EYFP-fused Y3. The gray curves drawn represent the best fits to the data.