Figure 4.

NFATc1 involved in chromatin remodeling mechanism. Chromatin remodeling complex proteins Brg-1 and Brm bind to the NFAT site in the c-myc promoter. (A) Nuclear extracts purified from MS, SUDHL-4, and OCI-LY10 DLBCL cells were analyzed for NFATc1, Brg-1, and Brm DNA binding to the NFAT consensus binding site by DNA-binding ELISA. IgG was used as a nonspecific control antibody. wt-CP, wild-type cold probe; mut-CP, mutant cold probe. (B) DLBCL MS nuclear extracts were subjected to gel-shift assay using the ³²P-labeled c-myc promoter NFAT DNA-binding site and antibodies to NFATc1, NFATc2, Brg-1, Brm, p65, and c-rel. wt-CP, wild-type cold probe; mut-CP, mutant cold probe. (C) Brg-1 interacts directly with NFATc1. DLBCL MS nuclear extracts (500 μg) or SUDHL-4 nuclear extracts (1 mg) were purified and subjected to coimmunoprecipitation analysis with IgG control or Brg-1 antibody. Eluted immunoprecipitated protein complexes were subjected to Western blotting for Brg-1 and NFATc1. (D) Colocalization of Brg-1 (green) and NFATc1 (red) in DLBCL MS and SUDHL-4 cells by confocal microscopy analysis. Colocalized areas appear yellow. Topro3, nuclear marker.