Fig. 5. PB interacts with EY in vitro and inhibits EY trans-activation activity. (A) The PB and EY proteins. HD, homeodomain; PD, paired domain. Grey boxes represent the conserved motifs (N, YPWM,C) of mammalian HOX-A2/B2 and PB (Cribbs et al., 1992). The region between amino acids 126-306 was fused to GST. (B) GST interaction assays. Aliquots (10 µl) of in vitro translated ³⁵S-methionine-labeled EY (lanes 1,5,12,16), EXD (lanes 3,7,9,13), EYA (lanes 4,8,10,14) and SO (lanes 2,6,11,15) were incubated with glutathione agarose beads containing bound GST (lanes 5-8), GST-PB (126-306; lanes 9-12) or GST-EN (lanes 13-16). An aliquot (5 µl) of in vitro translation products is shown in lanes (1-4). In this experiment, GST-EN bound 13% of the input ³⁵S-labeled EXD protein, whereas GST-PB bound 10% of input ³⁵S-labeled EY. (C) The interaction requires the EY PD and PB HD. GST `pull-down' assays were performed in the same conditions as in B, with ³⁵S-labeled full-length EY proteins (lanes 5,9) and truncated forms of EY lacking the HD (lanes 6,10), PD (lanes 7,11), or HD and PD (lanes 8,12), incubated with GST-PB (lanes 5-8) or GST-PB^sy (lanes 9-12), carrying the homeodomain R5H mutation indicated in A. Aliquots (5 µl) of in vitro translation products are shown in lanes 1-4. (D) Yeast one-and-a-half hybrid experiment. A strain carrying the integrated reporter vector pLacZi containing one copy of the so10 enhancer upstream of the minimal promoter of the yeast iso-1-cytochromeC gene. Vectors were pAS and pACT. pAS-EY, EY encoding vector. pACT-PB (126-306, see in A) is fused to the GAL4 activation domain. Lane 1, control with empty vector; lane 2, transactivation of the enhancer so10 with EY; lane 3, co-expression of EY and PB inhibits EY-dependent so10 transactivation, whereas PB does not activate so10 enhancer.