Ab128086

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).

ATRA/HL60 treated with various death-inducing stimuli as above, were incubated overnight at 37 °C. Supernatant was transferred to an eppendorf tube, centrifuged at 2,000 rpm for 2 minutes at room temperature, resuspended in 4 × sample buffer, boiled for 5 minutes, and centrifuged again at 14,000 rpm for 5 minutes. Then 25 ul were resolved by polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore). The membranes were blocked in 5 % non-fat milk in Tris-buffered saline (TBS) containing 0.1 % Tween 20, for 1 hour at room temperature. Membranes were probed with antibodies: monoclonal anti-citrullinated fibrinogen (Cayman Chemical, Ann Arbor, MI, USA), monoclonal anti-PAD2 antibody [10 (link)] (Abnova, #H00011240-M01, Taipei City, Taiwan), anti-PAD4 antibody [11 (link)] (Abcam #ab128086, Cambridge, MA, USA), monoclonal anti-fibrinogen (Abcam #ab10066), anti-citrullinated H3 (Abcam #ab5103), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Life Technologies #AM4300, Grand Island, NY, USA) and bound immunoglobulin was detected with horseradish peroxidase-linked secondary antibodies (Life Technologies). Reactivity was visualized with an ECL (enhanced chemilluminescence) substrate (Western Lightning® Plus-ECL, Perkin Elmer, Waltham Massachusetts) system.

Antibodies against Citrullinated Histone H3 (ab5103), PAD2 (ab16478), PAD4 (ab128086), PAD3 (ab183209), Tubulin and β-actin (ACTN05) were purchased from Abcam (Cambridge, UK). The anti-peptidyl-citrulline antibody, clone F95 (MABN328) was from EMD Millipore (Hertfordshire, UK). Antibodies against total histone H3 (1B1B2) and (96C10) were from Cell Signaling Technologies (Hertfordshire, UK). PAD1 (HPA062294) was from Sigma Aldrich (Dorset, UK), and PAD2 (12110-1-AP) was from Proteintech (Manchester, UK). All secondary antibodies used were raised in goat. F(ab')2 anti-Rabbit IgG A647, F(ab')2 anti-Mouse IgG PE (A10543), anti-Mouse IgG A594 (A11032) and anti-Mouse IgG A488 (A11029) were from Thermo Fisher Scientific (UK). Anti-Mouse IgM Texas Red was from VectorLabs (Peterborough, UK) and Anti-Rabbit IgG FITC was from Sigma Aldrich (Dorset, UK).
For flow cytometry, the following mouse anti human antibodies CD3 PEcy7, CD16 PerCP-Cy 5.5, CD19 BV786, CD56 BV650, and CD14 PE were obtained from BD Biosciences (Oxford, UK).

PAD4 (ab128086 and ab50247, Abcam), HA (MMS-101R, Covance), Flag (F3165, Sigma), AMC antibody kit (17-347, Millipore), E2F-1 (KH95 and C20, Santa Cruz Biotechnology), BRD4 (ab128874, Abcam), β-actin (A2228, Sigma), pRB (IF8, Santa Cruz Biotechnology), CD11B (ab75476, Abcam), mouse and rabbit secondary antibodies (GE Healthcare), and HA or Flag antibody-coupled agarose beads (Sigma) were used.

Cells were treated with the indicated compound or equal volumes of DMSO solvent 1 h before infection and throughout the entire duration of the infection as previously described in point 2.2. Cells were infected with HSV-1 at an MOI of 1 and harvested at the indicated time points. Cell lysates were prepared with RIPA buffer, quantified by Bradford method, and subjected to Western blot analysis. The primary antibodies were as follows: anti-PAD1 (Abcam, ab181791); anti-PAD2 (Cosmo Bio, SML-ROI002-EX); anti-PAD3 (Abcam, ab50246); anti-PAD4 (Abcam, ab128086); anti-PAD6 (Abcam ab16480); anti-actin (Sigma-Aldrich, A2066), anti-gD (Virusys, HA025-1), anti-ICP27 (Virusys, P1113), anti-IFIT1 (Invitrogen PA5-31254), anti-IFIT2 (Proteintech, 12604-1-AP), anti-IFIT3 (Proteintech 15201-1-AP).