Ac 40

For pupal wing westerns, 28-30 hr APF pupal wings were dissected directly into sample buffer. Two pupal wing equivalents were loaded per lane. Westerns were probed with affinity-purified rat anti-Pk [49 (link)], affinity purified rabbit anti-Dsh [49 (link)], rabbit anti-Stbm [50 (link)] and monoclonal mouse anti-actin (AC-40, Sigma, UK). A BioRad ChemiDoc XRS+ was used for imaging. Images were quantified using the Gel Analysis plug-in in ImageJ.

For the immunoblotting experiment, we used the following antibodies: rabbit monoclonal anti-PARP1 antibody (EPR18461: abcam, Cat#ab191217, 1/5000), mouse monoclonal anti-Actin antibody (AC-40: Sigma, Cat#A3853, 1/10000), rat monoclonal anti-α-tubulin antibody (YL1/2: Santa Cruz Biotechnology, Cat#sc-53029, 1/20000), rabbit polyclonal anti-PAR antibody (Enzo Life Science, ALX-210-890A-0100, 1/5000), rabbit polyclonal anti-NAMPT antibody (BETHYL, Cat#A300-372A, 1/10000), rabbit polyclonal anti-caspase-3 antibody (Cell Signaling, Cat#9662, 1/2000), mouse monoclonal anti-caspase-8 antibody (1C12: Cell Signaling, Cat#9746, 1/2000), mouse monoclonal anti-caspase-9 antibody (C9: Cell Signaling, Cat#9508, 1/2000), rabbit polyclonal anti-AIF antibody (Cell Signaling, Cat#4642, 1/5000), mouse monoclonal anti-Histone H2AX antibody (322105: R&D SYSTEMS, Cat#MAB3406, 1/1000) and rabbit polyclonal anti-γH2AX antibody (abcam, Cat#ab2893, 1/1000). We also used a horse anti-mouse IgG, HRP-linked antibody (Cell Signaling, Cat#7076); a goat anti-rabbit IgG, HRP-linked antibody (Cell Signaling, Cat#7074); and a goat anti-rat IgG, HRP-linked antibody (Cell Signaling, Cat#7077).

Whole cell extracts were made by lysing HeLa cells directly in boiling sodium dodecyl sulphate (SDS) sample buffer. Extracts were separated by SDS-polyacrylamide gel electrophoresis using precast 4–20% Precise protein gels (ThermoScientific). Separated proteins were transferred to PVDF-FL at 80V for 90 min at 4 °C. PVDF membranes were blocked in 5% milk/TBS 10% glycerol for 1 hr at RT, incubated in primary antibodies overnight at 4 °C, washed 3 × 20 min in TBS/0.05% TritonX-100 (TBS/T) before incubating in Roti block (Roth) containing secondary antibodies at 1 μg/ml – either anti-mouse DyLight 680 or anti-rabbit DyLight 800 (Cell Signalling). Membranes were incubated in the dark for 1 hr at RT in secondary antibodies. Membranes were washed 3 × 20 min in TBS/T, 1x in water for 5 min before visualising on the LiCor imaging system. Protein levels were quantified using Odyssey software and all protein levels were normalised to B-actin in order to compare individual wells.
Primary antibodies used for Western blotting are anti-Tpx2 (Novus NB500-179; 1:1000), B-actin (Sigma, AC40; 1:5000), anti-Y-tubulin (Sigma, GTU-88; 1:2000), anti-HSET (Santa Cruz, M-63; 1:1000), anti-ch-TOG (QED Bioscience, 34032, 1:1000).

The following reagents were used: rabbit anti-PICALM antibody (HPA019061, Sigma-Aldrich, 1:1000), mouse anti-actin antibody (AC-40, Sigma-Aldrich, 1:1000), rabbit anti-actin antibody (A2066, Sigma-Aldrich, 1:1000), rabbit anti-SREBP2 antibody (ab28482, 1:1000, Abcam, Cambridge, MA, USA,), IRDye680-conjugated donkey anti-rabbit antibody (926–68073, 1:5000, LI-COR, Biosciences, Lincoln, NB, USA), PE-conjugated mouse anti-human LDLR antibody (FAB2148P, 1:20, R&D Systems, Minneapolis, MN, USA), PE-conjugated mouse IgG Isotype antibody (IC002P, 1:20, R&D Systems).

Samples were harvested, washed in phosphate-buffered saline and after pelleting, lysed directly in lysis buffer (10 mm HEPES/KOH pH7.4, 10 mm KCl, 0.05% NP-40, 1 mm sodium orthovanadate). After centrifugation of the lysate, the supernatant containing the cytoplasmic components was retained, and the pelleted nuclei were washed with lysis buffer before extraction of the nucleoplasmic protein fraction in low salt lysis buffer (10 mM TRIS/HCl pH7.4, 0.2 mm MgCl₂). After blocking, membranes were probed with antibodies against actin (AC-40; Sigma), R26cit (ab19847; Abcam), R2,8,17cit (ab5103; Abcam), PADI2 (ab16478; Abcam) or PADI4 (ab128086; Abcam), followed by the appropriate horseradish peroxidase-linked secondary antibody (Cell Signalling, NEB, Hitchin, UK) and visualised using ECL Prime (GE Healthcare, Amersham, UK).

Whole-cell extracts were prepared by freeze and thaw lysis (three cycles) in 600 mM NaCl, 20 mM Tris-HCl pH 7.8, 20% glycerol. After SDS–PAGE, proteins were transferred onto PVDF membrane in semi-dry conditions. The membrane was then blocked in 5% non-fat dry milk buffer and incubated with mouse anti-γH2AX (Clone JBW301, Upstate, 1:5,000). Immunoblots were stained with corresponding HRP-conjugated secondary antibodies (GE Healthcare, 1:20,000) and detected with the enhanced chemiluminescence detection system (Amersham Biosciences). Quantification was performed using ImageJ.
For the validation of antibody specificity and cross-reactivity, a dilution series of synthetic peptides (CKATQASQEY; Peptide Specialty Laboratories GmbH), with the underlined serine in either phosphorylated or non-phosphorylated form, was immobilized on a nitrocellulose membrane at the indicated concentrations and probed with anti-γH2AX and anti-H2AX as described above.
CTCF knockdown western blots were developed using a rabbit anti-CTCF (#D31H2, Cell Signaling, 1:700) and a mouse anti-actin (AC-40, Sigma-Aldrich, 1:1,000) and overnight incubation at 4 °C, followed by a direct immunofluorescence detection using anti-rabbit-IgG-Cy5 (#711-175-152, Jackson, 1:1,000) and an anti-mouse-IgG-Alexa488 (A11029, Invitrogen, 1:1,000). Images were recorded using a AI600 Imager (Amersham) and quantified using ImageJ.