Fig. 4. Binding of Pax2/5/8 proteins to the proximal enhancer. (A) EMSA analysis of extracts prepared from dissected trunk or MHB tissue of 2-day-old and 3-day-old chick embryos (Pfeffer et al., 2000) with a 0.3 kb AvaI-ApaI probe of transgene #11 (Fig. 3A). In vitro synthesized Pax2b was used as a control. (B) Preferential binding of Pax2 and Pax5 to the proximal enhancer. Equimolar amounts of in vitro translated Pax proteins (quantitated by [³⁵S]Met incorporation) were analyzed by EMSA for binding to the 0.3 kb AvaI-ApaI probe. (C) Identification of a high-affinity Pax-binding site. Binding of Pax2b to the end-labeled S1 and S1m oligonucleotides was investigated by EMSA in the absence or presence of a 100-fold molar excess of the indicated oligonucleotides. (D) Alignment of the proximal enhancer sequences S1 and S2 with the consensus Pax2/5/8-binding site (Czerny and Busslinger, 1995). The S1m mutations are indicated.