Fig. 5. Biochemical analysis of BRO-1-RNT-1 interaction. (A) EMSA showing BRO-1-RNT-1 interaction. Lanes 1 and 4: GST-RNT-1 Runt domain (GST-RD) purified protein (20 ng) incubated with Runx consensus DNA binding site probe AACCGCA (10 fmol). A weak band shift can be observed (arrow) that is abolished by the addition of 30x (300 fmol); lane 2) or 100x (1 pmol; lane 3) of cold WT competitor. However, a mutated version of the competitor probe (AATCGAA), at 30x (lane 5) or 100x (lane 6), is also able to abolish RNT-1-DNA binding. The addition of purified BRO-1 protein (100 ng) to the reaction (lanes 7 and 10) causes the GST-RD band to be super-shifted and enhanced. These band shifts are still abolished by a cold WT competitor probe at 30x (lane 8) or 100x (lane 9), but this time the mutated competitor probe, added at 30x (lane 11) or 100x (lane 12), is unable to diminish RNT-1-DNA binding. (B) EMSA showing the RNT-1 e1241 point mutant I₁₁₂K does not interact with BRO-1. Lane 1, Runx consensus DNA binding site probe incubated with purified BRO-1 (1000 ng). No band shift is observed indicating that no non-specific DNA binding is occurring. Lane 2, 20 ng WT GST-RD incubated with Runx probe showing same band shift as in A. Increasing amounts of BRO-1 (lanes 3 and 4, 10 ng and 100 ng, respectively) causes the RNT-1-DNA band shift to be supershifted and enhanced as expected. Lane 5, 40 ng of GST-e1241 (Runt domain of RNT-1 containing the I₁₁₂K mutation found in the e1241 rnt-1 allele) incubated with Runx probe. Incubation with increasing amounts of BRO-1 (lanes 6 and 7, 10 ng and 100 ng, respectively) has no effect on the band shift, indicating that I₁₁₂K RNT-1 does not interact with BRO-1. (C) RUBY experiment showing direct interaction between BRO-1 and RNT-1 in mammalian cells. (i,iii,v) Fluorescence images. (ii,iv,vi) Merged fluorescence and phase contrast images. (i,ii) Cells expressing a RUBY-BRO-1 fusion. (iii,iv) Cells expressing a monomeric RUBY-RNT-1RD (Runt domain of RNT-1) fusion. (v,vi) Cells co-expressing both RUBY-BRO-1 and RUBY-RNT-1RD fusions.