Anti h4

The recombinant plasmids pET28a-His-CAPG and pGEX-4T-1-GST-PRMT5 were individually transformed into BL21(DE3) cells and incubated for 16 h on LB agar plates at 37 °C. Selected colonies were extracted and cultured in LB medium, followed by induction with 0.1 mM IPTG upon reaching an OD at 600 nm of 0.4. The transformed E. coli were lysed via sonication (VCX130/VCX130PB, Sonics & Materials, Inc., USA). The supernatant was collected after centrifugation at 10,000 ×g at 4 °C for 20 min. The supernatant was also passed through a Ni-NTA His-tag high-performance nickel-Sepharose column (GE Healthcare, Sweden). The GST and GST-tagged proteins were purified from bacterial lysates using glutathione-agarose beads (Amersham Biosciences). Purified core histone protein was purchased from Millipore (Millipore, Merck KGaA, Darmstadt, Germany).
Protein immunoprecipitation, GST pull-downs, and Western blotting were performed as described previously 57 (link), 60 (link), 61 (link). The following antibodies were used: anti-CAPG, anti-STC-1 (Santa Cruz Biotechnology, Inc., USA); mouse anti-FLAG (Sigma-Aldrich, USA); anti-PRMT5, rabbit anti-HA, mouse anti-HA, anti-His, rabbit anti-FLAG, anti-H3R8me2s, and anti-H4R3me2s (Abcam Inc., USA); anti-H4 (Millipore, Germany); and anti-GAPDH (ProteinTech Group, USA).

The following antibodies were used in this study: anti-HIRA (Active Motif, Carlsbad, CA, USA, WC119 and WC15), anti-Asf1a (Cell Signaling, Danvers, MA, USA, 2990), anti-Cabin1, anti-UBN1 (Abcam, Cambridge, MA, USA), anti-H3 (Abcam, ab1791), anti-H4 (Millipore, Billerica, MA, USA, 07–108), anti- H4S47p (Abcam), anti-H3.3 (Millipore, 09–838), anti-H3.1 and anti-H3.3 (provided by Dr Y Ohkawa), anti-pan-Akt, anti-Akt1, anti-Akt2, anti-p-Akt (S473) (Cell Signaling, 9271), anti-phospho-serine (Abcam, ab9332), anti-phospho-threonine (Abcam, ab9337), anti-myogenin (Santa Cruz, Santa Cruz, CA, USA, sc-576), anti-MHC (Upstate, Billerica, MA, USA, 05–716), 8WG16 (Covance, Princeton, NJ, USA, MMS-126R), anti-HA (Roche, Basel, Switzerland, 11 666 606 001), anti-Flag (Sigma-Aldrich, F3165), anti-Myc (Roche, 11 667 203 001), anti-β-catenin (Santa Cruz, sc-7963), and anti-α-tubulin (AbFrontier, Seoul, Korea, LF-PA0146). The anti-p-HIRA (Abmart, Shanghai, China) antibody was generated using the phospho-peptides RPRKD(pS)RLMPV and was affinity-purified from rabbit serum using a specific column. Antibody specificity was confirmed by checking the absence of cross-reactivity with the control peptide, RPRKDSRLMPV.