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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.

Human iPSC-CMs grown in 6-wells plates were harvested and lysed in RIPA buffer with Complete Mini, EDTA-free Protease inhibitor cocktail tablets (Roche). The lysates were placed on ice for 30 minutes, followed by centrifuging at 14000 rpm for 20 min, the supernatants were then collected as proteins. BCA Protein Assay kit (Thermo Fisher Scientific) was used to measure the protein concentration. Western blot was performed according to the standard protocol. Briefly, Equal amounts of protein was treated by SDSPAGE electrophoresis and transferred to a nitrocellulose membrane. After nonfat milk blocking, the membrane was incubated with the following primary antibodies at 4°C overnight, respectively: TLK1 (Fisher Scientific, 720397; 1:500 dilution), TEAD2 (Abcam, ab273017; 1:500 dilution), RBBP5 (Cell Signaling Technology, 12766S; 1:1000 dilution), ASH2L (Bethyl Laboratories, A300-107A-M; 1:500 dilution) and GAPDH (Abcam, ab8245; 1:1000 dilution). Subsequently, the membrane was incubated in protein-specific HRP conjugated secondary antibody for 1 hr at room temperature. The blots were visualized using chemiluminescence (Thermo Fisher Scientific).

The following antibodies were used: FLAG (Sigma, #F7425), KMT2D (Bethyl Laboratories, #A300-BL1185), UTX (Bethyl Laboratories, #A302–374A), ASH2L (Bethyl Laboratories, #A300–489A), WDR5 (Bethyl Laboratories, #A302–430A), DPY30 (Bethyl Laboratories, #A304–296A), NCOA6 (Bethyl Laboratories, #A300–411A), CUL1 (Invitrogen, #718700), FBXW7 (Bethyl Laboratories, #A301–720), C-MYC (Santa Crus, #sc-764), c-MYC-pT58 (Applied Biological Materials, #Y011034), HA (Biolegend, #901513), HA (Cell Signaling Technology, #3724), α-Tubulin (Santa Crus, #sc-8035), Skp1 (Santa Cruz, #sc-7163), CyclinE1 (Cell Signaling Technology, #4129), Cdk2 (Santa Cruz, #sc-6248), CyclinE (clone HE12), Vinculin (Santa Cruz, #sc-73614), ubiquitin K48 (EMD Millipore, 05–1307), Rabbit IgG-HRP (GE Healthcare, NA934V), mouse IgG-HRP (GE Healthcare, NA931V) and anti-FLAG M2 Affinity Gel (Sigma, #A2220).