Figure 7. Lin37 is not essential for DREAM assembly at promoters.
(A) DREAM components were purified from nuclear extracts of two independent density-arrested Lin37 knockout cell lines created by targeting exon 4 (KO-4) or exon 6 (KO-6) and NIH3T3 cells expressing Lin37 (WT). Purification was performed with a fragment of the mouse cyclin B2 promoter (Ccnb2) containing a CDE/CHR tandem element or a fragment of the Gapdhs promoter without CHR or E2F sites (Ctrl) to determine background binding. Protein binding was analyzed by Western blotting. All samples detected with one specific antibody were run on the same gel. (B) In vivo binding of DREAM to G2/M (blue) and G1/S (yellow) cell cycle gene promoters in the Lin37+/+ NIH3T3 parental cell line (WT) and a Lin37-/- cell line (Lin37-KO; clone 632-2) was analyzed by chromatin immunoprecipitation followed by semi-quantitative PCR (ChIP-qPCR). Enrichment of DREAM components normalized to input DNA is given. Non-targeting IgG and analysis of the non-DREAM binding Gapdhs promoter served as negative controls. Mean values ± SD of one representative experiment with three technical replicates are shown.