Fig. 2.

Scratch2 binds to the E-box of the GalR1 promoter. (A) The elution proteins from DNA pull-down assay were analyzed by mass spectrometry, and the resulting peptide of Scratch2 was identified. (B) A schematic structure of Scratch2. (C) Dig-labeled oligonucleotides were incubated with GST–Scratch2–ZF fusion protein, and the DNA–protein complexes were resolved by nondenaturing PAGE. (D) A schematic diagram for the GalR1 promoter harboring the E-box element. (E and F) PC12 cells (E) or vPAG tissues (F) were lysed, and ChIP assays were performed by using anti-Scratch2 antibody. Then, the immunoprecipitated DNA was detected by real-time PCR. In E, T₄ = 51.16, P < 0.0001. In F, T₄ = 18.39, P < 0.0001. (G) DNA affinity precipitation assays were performed with 293T cells extracts expressing Flag-Scracth2 and a biotin-labeled probe spanning −250 to −220 of the GalR1 promoter. DNA precipitates were detected with anti-Flag antibody. (H) The oligonucleotides described in Fig. 1F were labeled with digoxigenin. Dig-labeled oligonucleotides were incubated with GST–Scratch2–ZF fusion protein, and DNA–protein complexes were detected by nondenaturing PAGE. Data are presented as mean ± SD (n = 3 per group). ***P < 0.001.