Figure 5.

Inhibition of ADD1/SREBP1c DNA binding by Twist2 in vivo. (A) ChIP analysis of Twist2-mediated repression of the DNA-binding ability of ADD1/SREBP1c. H293 cells were transfected in duplicate (for use in western blot analysis) with the DNAs indicated. After cross-linking with formaldehyde, cells were lysed, pelleted and sonicated, and ADD1/SREBP1c was immunoprecipitated with anti-Myc antibody. Complexes were eluted, cross-linking was reversed and purified DNA was used as a template for PCR, using primers for the promoter region of ADD1/SREBP1c containing a SRE site. [α-³²P]dCTP-labeled PCR products were resolved by non-denaturing PAGE, and analyzed using a phosphoimager. (B) Western blot analysis of transfected proteins. Transfected cells were lysed, harvested and total protein was resolved by SDS–PAGE and evaluated using α-Myc and α-Flag antibodies to detect ADD1/SREBP1c and Twist2, respectively. Asterisk denotes a non-specific protein band.