Ab10158

Purified histones were electrophoresed in a 17.5% SDS polyacrylamide gel and transferred onto the polyvinylidene difluoride membrane at 15 V for 1 h by the semidry method. The membrane was blocked with TBS-T buffer containing 5% nonfat dried milk at room temperature for 1 h and then washed with TBS-T (twice for 10 min each). Membranes were incubated at 4 °C for 12 h with antibodies (from Millipore unless indicated otherwise) against H3 C-terminal region (07-690), H3K4ac (ABE223), H3K9ac (07-352), H3K14ac (Abcam, ab52946), H3K27ac (07-360), H3K36ac (07-540), H4 C-terminal region (Abcam, ab10158), H4K5ac (07-327), H4K8ac (07-329), H4K12ac (07-595), or H4K16ac (07-328). The membranes were then washed with TBS-T (4 times for 5 min each) and incubated with peroxidase-conjugated anti-mouse IgG (GE Healthcare, NA931) or anti-rabbit IgG (GE Healthcare, NA934) at room temperature for 1 h. The membranes were then washed with TBS-T (4 times for 5 min each) and were detected using enhanced chemiluminescence (Chemi-Lumi One Super: 02230-30).

To examine protein stability, exponentially growing cells were treated with 100 µg/ml of cycloheximide and collected. Whole-cell extracts were prepared as previously described in our studies [42 (link)]. Immunoprecipitation was performed as described [30 (link)] using the anti-Myc 9E10 (Covance) with protein G Sepharose, and myc- and FLAG-tagged proteins were detected with the anti-FLAG M2 (Sigma-Aldrich) and anti-Myc 9E10 antibodies, respectively.
For detection of histone H4 acetylation, whole-cell extracts were probed with the anti-acetyl-histone H4 antibody that recognizes histone H4 acetylated at the lysine 5, 8, 12, and 16 residues (06-088, EMD Millipore) as described [24 (link)]. Total levels of histone H4 were analyzed by the anti-histone H4 antibody (ab10158, Abcam).

Sperm pellet (1.8 × 10⁶ cells) was suspended in 180 μl of sperm lysis buffer [PBS containing 0.5% SDS, 10 mM DTT, 1 × complete protease inhibitors (Roche), and 1 mM PMSF]. After incubation for 30 min at 37 °C, sperm lysate was sonicated to shear DNA. For preparation of western blotting samples, 60 μl of 3 × sample buffer was added to 120 μl of lysate. DNA was purified from the remaining 60 μl of lysate, and the DNA amount was used as a loading control. Western blotting samples containing 3–30 × 10³ cells were applied to each well and western blotting analysis was performed. After blocking by PBS containing 3% BSA, the membrane was incubated with H3 antibody (1:3000–10,000 dilution; ab1791, Abcam) or H4 antibody (1:1000 dilution; ab10158, Abcam) and subsequently with peroxidase-conjugated anti-rabbit antibody (1:4000 dilution; Invitrogen). Chemical fluorescence signal was activated by ECL + (PerkinElmer), and the image was scanned with Odyssey systems (LI-COR). Recombinant proteins H3.1 (M2503S, NEB) and H3.3 (M2507S, NEB) were analyzed for checking sensitivities of H3 antibody to H3.1 and H3.3. Uncropped data of western blotting are provided in Supplementary Fig. 12.

ChIP assays were performed as described^{26 (link)}. Complexes were immunoprecipitated overnight with 2 μg of antibodies specific for SMYD3 (GTX121945, GeneTex), rabbit IgG (GTX35035, GeneTex), H3 (ab1791, Abcam, Cambridge, MA, USA), H3K4me3 (ab10158, Abcam), and RNA polymerase II CTD repeat YSPTSPS (phosphor Ser5) (ab5131, Abcam). Input samples were processed in parallel. Antibody/protein complexes were collected by 40 μl of protein G-coupled Sepharose beads (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA) and washed as follows: once with Tris/EDTA-150 mM NaCl, twice with Tris/EDTA-500 mM NaCl, and once with PBS. Immune complexes were eluted with 1% SDS and TE buffer. After decrosslinking, DNA was purified using a PCR cleanup kit (Qiagen) and analysed by qRT-PCR. The results were expressed as the percentage of the initial inputs. The primer sets used for the ChIP assay are listed in Supplementary Table S2.

The antibodies used in this study were anti-Myc antibody (sc-40, Santa Cruz), anti-HA antibody (sc-57592, Santa Cruz), anti-HBO1 antibody (sc-13284, Santa Cruz), anti-DDB2 antibody (sc-81246, Santa Cruz), anti-XPC antibody (sc-30156, Santa Cruz), anti-TFIIH p89 antibody (sc-293, Santa Cruz), anti-CPD antibody (NMDND001, Cosmo Bio), anti-Orc2 antibody (sc-13238, Santa Cruz), anti-acetyl-Histone H4 antibody (#06-866 Millipore), anti-Histone H4 antibody (ab10158, Abcam), anti-acetyl-Histone H3K14 antibody (A-4023, EPIGENTEK), anti-Histone H3 (tri methyl K4) antibody (ab1012, Abcam), anti-Histone H3 antibody (39763, ACTIVE MOTIF), anti-γH2AX antibody (50-171-736, Upstate), anti-SNF2H antibody (ab3749, Abcam), anti-ACF1 antibody (NB100-61041, Novusbio), anti-CSB antibody (553C5a, BIO MATRIX RESEARCH), anti-UVsS-A antibody (H00057654-B01, Abnova) and anti-β-actin antibody (sc-47778, Santa Cruz). Phospho Ser50 and Ser53 HBO1 rabbit polyclonal antibodies were generated by immunization with a synthetic phosphopeptide (CSARLpSQSpSQD). To perform immunoprecipitation of GFP-SNF2H, anti-GFP mAb-Agarose (D153-8, MBL) was used.

Tumor tissues for Western blots (~10 µg cell lysates) were acquired in a frozen or FFPE status following the tissue storage condition. Frozen tissues were homogenized using a lysis buffer (Cell Signaling, Danvers, MA, USA, #9803) with 1 mM PMSF (TCI, A2098) and protease inhibitor (Roche). The lysed homogenate was centrifuged and supernatants were collected and stored at −80 °C. FFPE tissues were placed in 1.5 mL tubes and deparaffinized in xylene at room temperature for 10 min. After the deparaffinization, tissues were rehydrated with a series of ethanol solutions (100, 90, 80, and 70%). Then, each tissue was resuspended in a cell signaling lysis buffer containing 1 mM PMSF and protease inhibitor. Each tissue sample was incubated on ice for 5 min and then at 80 °C for 2 h. After incubation, the supernatants were collected and stored at −80 °C.
Primary antibodies for HIST H1.5 (Abcam, ab18208, 1:1000), HIST H2A (Abcam, ab104075, 1:200), HIST H3 (Abcam, ab70550, 1:5000), HIST H4 (Abcam, ab10158, 1:1000), HMGA2 (Abcam, ab97276, 1:1000), and β-actin (Sigma-Aldrich, A5541, 1:10,000) were used, followed by horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG (1:5000) for 1 h and then visualized using the enhanced chemiluminescence detection system.