Fig. 5.

Protein-DNA interactions within the oNpt2a proximal promoter. (A) Shown is the −208 bp region of the 5’-flanking sequence of the opossum Npt2a gene [6]. The asterisks indicate the nucleotides that are 100% conserved between the opossum, mouse, and human Npt2a promoters. The TATA box transcription start site and potential binding elements for Sp1 and C/EBPβ are indicated (bold lettering and underlined). The arrow shows the position of −121 bp. (B-D) EMSA detection of protein-DNA interactions with the Sp1 and CAATT region of oNpt2a proximal promoter. B) Purified recombinant GST-NHERF-1 (*) and GST alone (G) were also tested for DNA binding. Nuclear extracts (NE) isolated from OK-WT (wt), OK-H (H), and OK-H - NHERF-1 stable transformant (NF) were incubated with radiolabeled DNA probes Sp1 or CAATT as indicated. Protein- DNA complexes were resolved by native electrophoresis and detected by autoradiography. Shown are representative images from 3 independent binding experiments using 2 separate NE isolations. (B) Specific complexes were identified by cold competition (see panel D) or antibody supershift (ss) (see panel C) and are indicated as I – II for Sp1 and I-IV for CAATT. Arrows indicate potential non-specific complexes. The sequences of the DNA probes are shown below the gel in panel B. (C) Protein interactions with the Sp1 probe. Binding reactions contained antibodies for either NHERF-1, C/EBPβ, or Sp3 as indicated. 250X, cold competition with 250-fold molar excess of Sp1 probe. ss, supershift. D) Protein interactions with the C/EBP probe. Binding reactions contained antibodies for either NHERF-1 or C/EBPβ as indicated. 250X, cold competition with 250-fold molar excess of C/EBP probe.