Ab1790

Cells were lysed in Laemmli sample buffer (Laemmli, 1970 (link)) supplemented with 10% 2-mercaptoethanol (133-1457; Wako) and incubated at 95°C for 5 min to denature proteins. Then, SDS-PAGE was performed to separate the proteins. Proteins in the gel were transferred to an Immobilon-P membrane (IPVH00010; Merck) and blocked with PBS-T containing 5% nonfat milk (190-12865; Wako) for 30 min at room temperature. Subsequently, the proteins were blotted with antibodies at the indicated dilutions: rabbit anti–histone H2B (ab1790; Abcam) at 1:10,000 or 1:20,000; rabbit anti–HaloTag (G9281; Promega) at 1:1,000; mouse anti–CTD of RPB1 (ab817; Abcam) at 1:1,000; mouse anti–α-Tubuline (T6199; Sigma-Aldrich) at 1:5,000; mouse anti–CDK9 (sc-13130; Santa Cruz) at 1:500; horseradish peroxidase-linked goat anti–rabbit IgG whole antibody (170-6515; Bio-Rad) at 1:5,000 for anti-H2B and anti-HaloTag; HRP-linked goat anti–mouse IgG whole antibody (170-6516; Bio-Rad) at 1:5,000 for anti-CTD, anti–α-Tubuline, and anti-CDK9. Signal detection was performed by using the Immobilon Western Chemiluminescent HRP substrates (WBKLS0500; Merck) with a chemiluminescence CCD imaging system EZ-Capture MG (ATTO).

To resolve nuclear multiprotein complexes, nuclear extracts from S1 cells (23 (link)) were loaded onto a 10–40% sucrose gradient and ultracentrifuged for 40 h at 4°C and 214 000 g. Fractions of equal volumes were precipitated with trichloroacetic acid and analyzed with the pellet (insoluble fraction) by western blot. In immunoprecipitation (IP) experiments, nuclear extracts (1 mg) were incubated with antibodies overnight at 4°C and further processed using the Universal Magnetic Co-IP kit (Active Motif) according to the manufacturer's instructions. Antibodies used for immunoblotting were: 53BP1 (Abcam, Ab36823, 1 μg/ml), BRCA1 (Calbiochem, MS110, 5 μg/ml), BRG1 (Milipore, 07–478, 1:10000), DNA-PKcs (Abcam, clone 18–2, 2 μg/ml), γH2AX (Ser139; Millipore, clone JBW301, 1 μg/ml), Histone H2B (Abcam, Ab1790, 0.1 μg/ml), lamin B (Abcam, Ab16048, 60 ng/ml), NuMA (B1C11, 1:2, a gift from Dr Jeffrey Nickerson, UMass, Worcester, USA), PAR (Trevigen, 4336-APC-050, 1:1000), phospho-NuMA (Cell Signaling, 1:1000), SNF2h (Abcam, Ab3749, 1 μg/ml), RAD51 (Abcam, Ab63801, 1:1000), tubulin (Abcam, Ab3194, 1 μg/ml) and Williams Syndrome Transcription Factor (WSTF; Cell Signaling, 0.3 μg/ml). For IP: NuMA (Oncogene, clone Ab-2 or Bethyl Laboratories) and SNF2h (Abcam, clone 3.25(2)).

The antibodies used in this work were against: H3 (Abcam, ab1791, dilution 1/2000), H2B (Abcam, ab1790, dilution 1/2000), phosphorylated CHK1 (Cell Signaling, 2341 S, dilution 1/250), PCNA (Sigma, P8825, dilution 1/2500), RPA34^{62 (link)} (dilution 1/500), MCM3^{61 (link)} (dilution 1/2000), CDC45^{63 (link)} (dilution 1/1000), ELYS^{31 (link),64 (link)} (dilution 1/500), MCM4^{63 (link)} (dilution 1/1000), anti-Chk1 (dilution 1/500), anti-ORC5 (dilution 1/1000), anti-CDC6^{63 (link)} (dilution 1/500), OCT4 (Abcam, ab19857, dilution 1/500), actin (Sigma, A4700, dilution 1/500), HRP-linked ECL anti-mouse IgG (GE Healthcare, NA931V, dilution 1/4000), HRP-linked ECL anti-rabbit IgG (GE Healthcare, NA934V, dilution 1/4000) (For details see Supplementary Table 7).

Chromatin samples were fractionated on a 4–14% SDS-PAGE gel, transferred to nitrocellulose membrane using Towbin buffer (25 mM Tris pH 8.8, 192 mM Glycine, 20% methanol) on a Hoefer TE 77 semi-dry transfer unit for 2 h at 55 mA/membrane. The membrane was incubated in blocking solution (5% non-fat milk in TBS) for 1 h prior to replacement with primary antibody diluted in TBS with 1.5% non-fat milk: anti-Histone H3 (abcam ab1791, 1:10,000), anti-Histone H2B (abcam ab1790, 1:1000), anti-Histone H2A (abcam ab18255, 1:1000), anti-Histone H4 (abcam ab10158, 1:1000), anti-Histone H3K18ac (Active Motif 39,755, 0.5 μg/ml) or anti-Histone H3K27me2 (Active Motif 39,245, 1:1000). Membrane was incubated on a rocker overnight at 4 °C, washed in TBS-T (TBS, 0.1% Tween-20) three times for 10 min and incubated in TBS with 1.5% non-fat milk containing a secondary goat anti-rabbit IgG Alexa Fluor conjugated antibody (Life Technologies A27042, 1:15,000) for 45 min at room temperature. The membrane was washed in TBS-T three times for 10 min, rinsed in diH₂O and visualized using a LI-COR Odyssey. Membrane was then stained with 0.1% amido black 10B (Sigma N3393) in 10% acetic acid for 1 min, destained with 5% acetic acid twice for 1 min and rinsed in diH₂0 twice for 10 min before imaging using a LI-COR Odyssey.