Samples were immediately subjected to formaldehyde crosslinking (final concentration, 1%), and chromatin was isolated according to a method previously described (Durand-Dubief and Ekwall 2009 (link)). Antibodies against H4K20me1 (Abcam ab9051), H4K20me2 (Abcam ab9052), H4K20me3 (Abcam ab9053), HA (Abcam ab9110), T7 (EMD chemicals 69522), H3cter (Abcam ab1791), and H4 Pan (Millipore 05-858, clone 62-141-13) were used.
Ab9052
Manufactured by Abcam
Sourced in United States
Ab9052 is a primary antibody targeting the enzyme Tyrosine-protein kinase SYK. It is suitable for use in various applications, including Western Blotting, Immunohistochemistry, and Immunocytochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Ab9051, by Abcam (7 mentions)
Ab9053, by Abcam (4 mentions)
Ab1791, by Abcam (4 mentions)
Ab10158, by Abcam (3 mentions)
Ab1791, by Merck Group (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Ab8898, by Abcam (2 mentions)
Secondary horseradish peroxidase coupled antibodies, by Vector Laboratories (2 mentions)
H4k20me1, by Abcam (1 mentions)
Sc 6954, by Santa Cruz Biotechnology (1 mentions)
Sc 271682, by Santa Cruz Biotechnology (1 mentions)
Alexa fluorophore, by Thermo Fisher Scientific (1 mentions)
F1804, by Merck Group (1 mentions)
Anti g9a, by Merck Group (1 mentions)
Gtx70103, by GeneTex (1 mentions)
C1303, by Applygen (1 mentions)
Anti dna pkcs, by Santa Cruz Biotechnology (1 mentions)
Mab3802, by Merck Group (1 mentions)
Anti actin, by Santa Cruz Biotechnology (1 mentions)
Ab2175, by Abcam (1 mentions)
13 protocols using ab9052
1
Chromatin Isolation and Antibody Binding
2
Immunostaining for DNA Damage Response
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Cells were grown on sterile 12 mm glass coverslips, fixed in 3% formaldehyde in PBS for 15 min at room temperature, washed once in PBS, permeabilized for 5 min at room temperature in PBS supplemented with 0.2% Triton X-100 (Sigma-Aldrich), and washed twice in PBS. All primary and secondary antibodies (Alexa fluorophores; Life Technologies) were diluted in filtered DMEM containing 10% FBS and 0.02% Sodium Azide. Antibody incubations were performed for 2 h at room temperature. After antibody incubations, coverslips were washed once with PBS and incubated for 10 min with PBS containing 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI, 0.5 μg/ml) at room temperature to stain DNA. After three washing steps in PBS, coverslips were briefly washed with distilled water and mounted on 6 μl Mowiol-based mounting media. The following primary antibodies were used for immunostaining: H2AX Phospho S139 (mouse, 613401, 1:1,000; BioLegend), 53BP1 (mouse, Upstate MAB3802, 1:1,000), H4K20me2 (rabbit, ab9052, 1:100; Abcam), H4K20me1 (rabbit, ab9051, 1:200; Abcam), BRCA1 (mouse, sc-6954, 1:100; Santa Cruz), Cyclin A (mouse, sc-271682, 1:100; Santa Cruz), and RAD51 (rabbit, 70-002, 1:1,000; Bioacademia).
3
Antibody Validation for Chromatin Research
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The antibodies used in this study were: anti-His-tag (D291-3, MBL, Aichi, Japan), anti-Myc-tag (M047-3, MBL), anti-GFP-tag (M048-3, MBL), anti-GLP (D220-3, MBL), anti-Flag-tag (F1804, Sigma-Aldrich, St Louis, MO, USA), anti-G9a (G6919, Sigma-Aldrich), anti-GST-tag (C1303, APPLYGEN, Beijing, China), anti-mCherry-tag (C1329, APPLYGEN, Beijing, China), anti-H3 (ab1791, Abcam, Cambridge, UK), anti-H4 (ab10158, Abcam), anti-H3K9me2 (ab1220, Abcam), anti-H3K27me3 (ab6002, Abcam), anti-H3K9me3 (ab8898, Abcam), anti-H3K79me1 (ab2886, Abcam), anti-H4K20me1 (ab9051, Abcam), anti-H4K20me2 (ab9052, Abcam), anti-MRE11 (ab12159, Abcam), anti-RPA32 (ab2175, Abcam), anti-phospho-Histone H2AX (Ser139) (05–636, EMD Millipore, Billerica, MA, USA), anti-53BP1 (MAB3802, EMD Millipore), anti-GLP (09–078, EMD Millipore), anti-FK2 (04–263, EMD Millipore), anti-53BP1 (NB100–304, Novus Biologicals, Abingdon, UK), anti-GLP (B0422, Novus Biologicals), anti-DNA-PKcs (sc-1552, Santa Cruz Biotechnology), anti-ATR (sc-1887, Santa Cruz Biotechnology), anti-Actin (sc-58673, Santa Cruz Biotechnology), anti-SET8 (C18B7, Cell Signaling Technology, Danvers, MA, USA), anti-ATM (GTX70103, GeneTex), anti-RNF8 (14112-1-AP, Proteintech, Wuhan, Hubei, China) and anti-RNF168 (21393-1-AP, Proteintech).
4
Nuclear Protein Levels Analysis after PM10 and BaP Exposure
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The nuclear protein levels of H4K20me2 and XPA Ser196 were evaluated after the A549 cells were exposed to PM10 and BaP for 24 h. Protein extraction was performed by separating the nuclear protein fraction and cytoplasmic protein fraction using Chemicon’s nuclear extraction kit (Millipore, 2900, Billerica, MA, USA) according to the manufacturer’s instructions. Protein quantification was performed using the bicinchoninic acid assay, as previously mentioned. Fifteen micrograms of nuclear protein fraction was loaded into a 15% SDS-polyacrylamide gel, and the levels of proteins were determined as previously described. Anti-H4K20me2 antibody (Abcam, ab9052) at 1:2000 and anti-phospho-XPA (Ser196) antibody (Thermo Fisher, 64730) at 1:500 were incubated overnight at 4 °C under constant agitation. Histone 3 (H3) (Abcam, ab1791) was used as a housekeeping protein at 1:5000 for 1 h at room temperature, followed by the incubation of HRP-secondary anti-mouse antibody (Amersham, NA931).
5
Histone Modification Detection by Western Blot
6
Antibody Validation and Characterization
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The antibodies used in our study are: anti-HA (H3663, 1:2000 dilution) from Sigma; anti-H3 (ab1791, 1:5000 dilution), anti-H3K9me3 (ab8898, 1:2000 dilution), anti-H3K4me3 (ab8580, 1:2000 dilution), anti-H3K36me2 (ab9049, 1:2000 dilution), anti-H3K36me3 (ab9050, 1:2000 dilution), anti-H4K20me2 (ab9052, 1:2000 dilution) and anti-H4K20me3 (ab9053, 1:2000 dilution) from Abcam; anti-H1 (pAb)(61202, 1:2000 dilution) from Active Motif; anti-H3K27me3 (9733S, 1:2000 dilution) and anti-SUZ12 (3737S, 1:2000 dilution) from CST; anti-H3K9me2 (NB21-1072S, 1:2000 dilution) from NOVUS and anti-H3K4me2 (07-030, 1:2000 dilution) from Millipore; anti-V5 (P01L075, 1:2000 dilution) from Gene-Protein Link. The uncropped and unprocessed scans of all of the blots are shown in the Source Data file.
7
Immunofluorescence detection of histone modifications
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For immunofluorescence experiments 2 × 103 cells were seeded onto 8-well Lab-Tek chamber slides (Thermo Scientific, 177445) and grown for 48 h. Samples were fixed with 4% formaldehyde (252931 Panreac) for 15 min at RT, and permeabilized with 1 × PBS/0.1% Triton X-100 (Sigma-Aldrich) for 20 min at RT. Blocking was performed with 1 × PBS/10% BSA (A7906 Sigma-Aldrich) at RT for 1 h, followed by incubation with the corresponding primary antibody _ rabbit anti-H4K20me1 (ab9051, Abcam) or rabbit anti-H4K20me2 (ab9052, Abcam)_ at 1:1,000 dilution (see Supplementary Table 2 ) in antibody diluent (EnVision FLEX DM830 Dako), for 1 h at 4°C. Secondary antibody chicken anti-rabbit IgG Alexa Fluor 488 (A-21441 Invitrogen) at 1:500 dilution was incubated for 1 h at RT protected from light. Finally, slides were mounted using EverBrite mounting medium with DAPI (23002 Biotium). Immunofluorescence images were acquired with a Zeiss microscope, equipped with a 63X/1.4 NA immersion objective and an AxioCam MRm camera (Carl Zeiss). Fluorescence intensity measurements were performed using the ZEN lite software (ZEN lite 2.3 SP1, Carl Zeiss).
8
Monoclonal Antibody Generation and Immunoblotting
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The monoclonal antibodies against CBLL1 (Y6018 and Y6037, 430–491 amino acids were used as an immunogen) and VIRMA (Y1639, 1421–1480 amino acids were used as an immunogen) were generated using a baculoviral display system, as described previously (66 (link)). Polyclonal anti-human WTAP antibodies raised in rabbits were used for immunoblotting (16 (link)). The following antibodies were used for immunoblot analysis: ZC3H13 (ab70802, Abcam), BCLAF1 (A300-608A, Bethyl), THRAP3 (A300-956A, Bethyl), CBLL1 (ARP39623_T100, Aviva Systems Biology), RBM15 (ab70549, Abcam), RBM15B(1C2C11, Proteintech), METTL3 (D2I6O, Cell Signaling Technology), METTL14(ab220030, Abcam), H4K20me1 (ab9051, Abcam), H4K20me2 (ab9052, Abcam), H4K20me3 (ab9053, Abcam), beta-Actin (A5441, Sigma), V5(MA5-15253, Invitrogen) and FLAG(F7425, Sigma Aldrich).
9
Demethylation Assay of Nucleosomes
10
Quantifying H4K20me2 Genomic Enrichment
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For real-time PCR quantification of H4K20me2-enriched genomic regions (qChIP), LN-229 cells (empty vector, KMT5B #3, and KMT5B #7) were freshly processed using the SimpleChIP Enzymatic Chromatin IP Kit with magnetic beads (Cell Signaling, 9003). Immunoprecipitations were performed using antibodies against H4K20me2 (ab9052, Abcam), total histone H3 (Abcam, ab1791) as positive control, and IgG antiserum (Abcam, ab46540) as negative control (see Supplementary Table 2 ). DNA was purified and used for quantitative real-time PCR with SYBR Green 2X PCR Master Mix (Applied Biosystems) and the primers for the gene loci listed in Supplementary Table 1 . Input chromatin DNA was used to create a standard curve and determine the efficiency of amplification for each primer set in a StepOnePlusTM Real-Time PCR machine (Applied Biosystems). All samples were measured in triplicate. IgG was used as negative control. ChIP data were analyzed and are shown in the results as the percentage relative to the input DNA amount by the equation:
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