Figure 4.

Inhibition by H89 of TPA-induced nucleosomal response and chromatin remodeler/modifier recruitment to the Jun, Cox-2 and Fosl1 5′ proximal regulatory regions. (A) Serum-starved 10T1/2 cells were pre-treated or not with H89 prior to TPA stimulation for 0, 15 or 30 min. Formaldehyde crosslinked mononucleosomes were prepared and used in ChIP assays with antibodies against MSK1, H3S10ph, H3S28ph, 14-3-3ε, 14-3-3ζ, BRG1, PCAF or H3K9acK14ac. Equal amounts of input and immunoprecipitated DNAs were quantified by real-time quantitative PCR. The enrichment values of the 5′ proximal sequences of Jun (−146), Cox-2 (−111) and Fosl1 (−187) genes are the mean of three independent experiments, and the error bars represent the standard deviation. (B) Serum-starved 10T1/2 cells were pre-treated with H89 prior to TPA stimulation for 0 or 30 min. Aliquots of 1 mg total cell extract were incubated with anti-MSK1 antibodies. The whole ‘Bound’ and ‘Non-specific’ fractions and equivalent volumes of ‘Input’ and ‘Un-bound’ fractions, corresponding to 25 μg of total cell extracts were resolved on SDS–10%-PAGE and immunoblotted with indicated antibodies (IB).