Anti h4k16ac

Initial assessment of sgRNA activity was performed by transducing individual sgRNAs into fibroblasts containing a doxycycline-inducible Cas9. Cells (2 × 10⁶) were plated in 10 cm dishes and infected with pLenti_BSD_sgRNA lentivirus for 24 h. Transduced cells were selected with blasticidin for 4 days, after which Cas9 was induced for 1–2 weeks. KO was assessed via FACS, quantitative immunofluorescence microscopy, and/or TOPO-cloning and Sanger sequencing of sgRNA target genes. For TOPO sequencing, primers were designed to amplify genomic DNA sequences surrounding predicted sgRNA target sites (Supplementary Table 4). PCR was performed on genomic DNA from cells expressing Cas9 and respective sgRNAs, and amplicons cloned into blunt-end TOPO vectors. Resultant TOPO-vectors were transformed into Stbl3 competent E. coli, and DNA extracted for sequencing. Standard M13 sequencing primers were used to sequence the cloned amplicons. For FACS analysis a cell line transduced with empty pTRIPZ (Open Biosystems) and expressing TurboRFP was used. FACS analysis and immunofluorescence analysis was performed as previously described [38 (link)]. Antibodies used were anti-H1.0 (Upstate) and anti-H4K16Ac (Cell Signaling Technology). Quantification of fluorescence intensity was performed using Metamorph software.

The antibodies anti-H3K9ac (#2594), anti-H3K27ac (#8173), anti-H3K56ac (#07-677-1S), anti-H4K8ac (#2594), anti-H4K12ac (#13944), anti-H4K16ac (#13534), anti-H3 (#4499), anti-H4 (#13919), anti-BRD4 (#13440), anti-Tom20 (#42406), anti-γ-H2AX (#80312), anti-H2AX (#7631), anti-p-TBK1(Cell #5483), anti-TBK1 (#3504), anti-p-IRF3 (#29047y), anti-IRF3 (#11904), anti-p-P65 (#3033), anti-P65 (#8242), anti-p-STAT1(#9167), anti-STAT1(#9172), anti-STING (#3337) and anti-p-P53 (#9286) were from Cell Signaling Technology; anti-cGAS (26416–1-AP), anti-P53 (10442-1-AP), anti-MDM2 (19058-1-AP) and anti-P21 (#10355-1-AP) were from Proteintech; anti-dsDNA (MAB1293) was from Millipore; and anti-actin (A1978) was from Sigma. Antiserum against PRV gE was generated by immunization of mice with purified recombinant gE.

The antibodies used in the present work were as follows: anti‐Flag (F3165, Sigma‐Aldrich), anti‐Flag@M2 Affinity Gel(AZ220, Sigma), anti‐TIP60 (sc‐166323, Santa Cruz Biotechnology), anti‐SENP3 (ab124790, Abcam), anti‐DNA‐PKcs (ab32556, Abcam), anti‐DNA‐PKcs pT2609 (ab97611) and pS2056 (ab18192) (both Abcam), anti‐ATM (sc‐135663, Santa Cruz Biotechnology), anti‐ATM pS1981 (ab81292, Abcam), anti‐HA (H9658, Sigma), anti‐acetylated‐lysine antibody (9441s, Cell Signaling Technology), anti‐histone H4 (2592) and anti‐CHK2 pT68 (2661) (both Cell Signaling Technology), anti‐H4K16ac (13534s, Cell Signaling Technology), anti‐CHK1 (sc‐8408, Santa Cruz Biotechnology), anti‐CHK1 pS345 (2348s, Cell Signaling Technology), anti‐CHK2 (ab109413, Abcam), anti‐β‐actin (TA‐09, ZSGB‐BIO), anti‐γH2AX(05‐636, EMD Millipore), Alexa Fluor 488‐labelled Goat Anti‐Mouse IgG(H+L)(A‐21202, Invitrogen), Alexa Fluor 568‐labelled Goat Anti‐ Rabbit IgG(H+L) (A‐11036, Invitrogen). Cisplatin (PHR1624), etoposide (E1383), campathecin (C9911) and mitomycin C (M0503) were purchased from Sigma‐Aldrich. Annexin V, FITC Apoptosis Detection Kit (AD10) was purchased from DOjindo.