Med23

The preparation of chromatinized G5E4T, normalization of chromatin amounts, in vitro transcription reactions on chromatin templates, immunodepletion and IT procedures have been described in our previous publications (Black et al., 2006 (link); Lin et al., 2011 (link)). Briefly, transcription or IT assays were carried out on 50 ng of chromatin templates that were bound with saturating levels of GAL4-VP16. Where indicated, immobilized chromatin was acetylated with 20 ng of Esa1, 20 ng Gcn5 and 1 mM Acetyl-CoA for 45 min. Transcription was measured by ³²P-primer extension and visualized on an Amersham Biosciences Typhoon 9400. Immunoblots were processed on a LI-COR Odyssey and quantitated by Image Studio 2. Antibodies included pan-acetyl Histone H3 (Active Motif), MED23 (BD Pharmingen), Pol II (QED Bioscience), TFIIB (Tantin et al., 1996), EP400, Tip60 (Abcam), ASH2L, CHD1 (Bethyl Laboratories) and all others from Santa Cruz Biotechnology.

Nuclear proteins were isolated from patient and control skin fibroblasts according to the instructions of the Qproteome Cell Compartment Kit (Qiagen, Valencia, CA). Proteins were then resolved by SDS-PAGE and transferred to a nitrocellulose membrane. The blot was incubated with antibodies against MED23 (BD Pharmingen, San Jose, CA) and histone H2B (EMD Millipore, Billerica, MA) and visualized using the Odyssey CLx infrared imaging system (LI-COR Biosciences, Lincoln, NE).