Fig. 3. Efficient ARF2 competition with ARF1 is explained by the high affinity SIM in XFast-3. (A) XFast-3 competes very efficiently with XFast-1 for binding activated Smads and the ARE. NIH3T3 cells were transfected with 1 µg HA-tagged XFast-1 alone or together with 0.25, 0.5, 0.75 or 1 µg HA-tagged XFast-3 or with 1 µg of XFast-3 and the same titration series of HA-tagged XFast-1. Cells were induced (or not) with TGFß1 as indicated. Whole-cell extracts were prepared and analysed for ARF1 and ARF2 by bandshift on the ARE (upper panel) or western blotted with anti-HA antibody (lower panel). ARF1 and ARF2 are indicated, as is the complex of XFast-1 with DNA. No XFast-3-DNA complex is detected. (B,C) A peptide corresponding to the XFast-3 SIM disrupts ARF1 and ARF2 much more efficiently than a peptide corresponding to the XFast-1 SIM. NIH3T3 cells were transfected with Flag-tagged XFast-1 (B) or XFast-3 (C) and incubated with TGFß1 to induce formation of ARF1 or ARF2, respectively, in a bandshift assay on the ARE. Increasing amounts (pmoles) of wild-type (wt) or mutant (mut) SIM peptides corresponding to the SIMs of XFast-3 or XFast-1 were included in the bandshift reactions as indicated.