Supplemental Material

This article contains the following supporting material:

• Figure 1 - Construction of Brd4+/ - clones. (A) Schematic representation of targeting strategy. The positions of the puromycin resistant gene and Brd4 exons (3, 4 and 5) are shown. The external probe is a unique 3’ genomic sequence that distinguishes the wild type 12 kb BamHI fragment from a 7.5kb BamHI fragment generated by the targeted allele. (B) Southern blot analysis of genomic DNA obtained from selected ES clones.
• Figure 2 - The cell growth rate and mitotic indices of Brd4+/ - cells. (A) One x 10⁵ cells of Brd4+/ + (WT) and three Brd4 +/ - clones were placed in 4 ml of media and passed every two days for 8 days, and the number of cells were estimated on the indicated days. The values represent the average of three experiments. (B) Brd4+/ + and +/ - cells were incubated with nocodazole at 100 ng/ml for indicated periods of time (h). Mitotic cells were scored after staining of DNA and counted. The values represent the average of three tests.
• Figure 3 - Solubility of Brd4 in G₁ cells following nocodazole removal. Brd4 +/ + and +/ - cells were treated with nocodazole (100 ng/ml) for 8 h. Mitotic cells were incubated in fresh media for 90 min allowing to proceed to G1 phase. Cells were lysed and nuclear pellets were prepared as described (Dey et al., 2003). Proteins were extracted with indicated concentrations of NaCl and resolved on SDS-PAGE and immunoblotted for Brd4 and TFIIB. Proteins present in the soluble and insoluble fraction are shown. TFIIB was used as an internal control.