DNA-binding ability of Pax2 revealed by electrophoretic mobility shift assays.
(A) Western blot analysis of recombinant Pax2 protein with a V5 tag at its C terminus purified by affinity chromatography. The purified Pax2 protein is detected by anti-V5-HRP antibody (lane 2). E. coli lysate with the vector only (pET101/D-TOPO) was used as a negative control and no detection was observed for this control with anti-V5-HRP antibody (lane 1). (B) Detection of Pax2 binding sites. Electrophoretic mobility shift assays were performed using purified Pax2 and the 32P-end-lableled oligonucleotide probe cwp1-45/−1 (−45 to −1 relative to the translation start site of the cwp1 gene). Components in the binding reaction mixtures are indicated above the lanes. The Pax2 binding specificity for the cwp1-45/−1 probe was confirmed by competition and supershift assays. Some reaction mixtures contained 200-fold molar excess of cold oligonucleotides cwp1-45/−1 or ran-30/−1 or 0.8 µg of anti-V5 antibody, as indicated above the lanes. The arrowhead indicates the shifted complex. The transcription start site of the cwp1 gene determined from 24-h encysting cells is indicated by an asterisk . The AT-rich Inr element spanning the transcription start site is underlined.