Cells were plated in 15-cm dishes 24 h before treatment with 0.1% dimethylsulfoxide or compound (6-point, fivefold serial dilution). Cells were lysed with 4% SDS and homogenized using QIAshredder columns (Qiagen). Proteins were separated on 4–12% Bis-Tris gels (Invitrogen) and transferred to nitrocellulose membranes (Invitrogen). Membranes were blocked with 5% milk and probed with primary antibodies against DNMT1 (1:1,000, Cell Signaling Technology, 5032), DNMT3A (1:1,000, Cell Signaling Technology, 3598), DNMT3B (1:1,000, Cell Signaling Technology, 67259), Phospho-Histone H2A.X (1:1,000, Cell Signaling Technology, 9718), Histone H2A.X (1:1,000, Cell Signaling Technology, 2595) or Vinculin (1:100,000, Sigma, SAB4200080). After washing, membranes were probed with IRDye secondary antibodies (LI-COR), washed again and imaged using a LI-COR Odyssey CLx Imager.
Histone h2a x
Manufactured by Cell Signaling Technology
Sourced in United States
Histone H2A.X is a core histone protein that is a variant of the H2A histone family. It is involved in the cellular response to DNA double-strand breaks.
Automatically generated - may contain errors
Lab products found in correlation
γh2ax, by Cell Signaling Technology (3 mentions)
β actin, by Merck Group (3 mentions)
Phospho histone h2a x, by Cell Signaling Technology (2 mentions)
Dnmt3a, by Cell Signaling Technology (2 mentions)
Dnmt1, by Cell Signaling Technology (2 mentions)
E cadherin, by Santa Cruz Biotechnology (2 mentions)
Chemidoc xrs system, by Bio-Rad (2 mentions)
Odyssey clx imager, by LI COR (1 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (1 mentions)
Irdye secondary antibody, by LI COR (1 mentions)
Qiashredder column, by Qiagen (1 mentions)
Vinculin, by Merck Group (1 mentions)
Bis tris gel, by Thermo Fisher Scientific (1 mentions)
Dnmt3b, by Cell Signaling Technology (1 mentions)
Protease and phosphatase inhibitor, by Roche (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Amersham imager 600rgb detection system, by GE Healthcare (1 mentions)
Stat3, by ABclonal (1 mentions)
Phospho p65, by Cell Signaling Technology (1 mentions)
11 protocols using histone h2a x
1
Quantifying DNA Methylation Regulators
2
Colon Tissue Protein Extraction and Analysis
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Colon tissue proteins were extracted with RIPA buffer (Beyotime) supplemented with protease and phosphatase inhibitors (Roche). Whole-tissue lysates were loaded and subjected to SDS-PAGE, transferred onto polyvinylidene difluoride (PVDF) membrane (Millipore), and then blotted by the Amersham Imager 600RGB detection system (GE Healthcare), as described previously [22 (link)]. Antibodies used are as follows: phospho-STAT3 (Cell Signaling Technology, 9145), phospho-P65 (Cell Signaling Technology, 3033), P65 (Cell Signaling Technology, 8242), phospho-Histone H2A.X (Cell Signaling Technology, 9718), Histone H2A.X (Cell Signaling Technology, 7631), STAT3 (Abclonal, A19566,), TIPE (Proteintech, 15790-1-AP,) and β-actin (Proteintech, 66009-1-Ig).
3
Comprehensive Protein Expression Analysis
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All cells were harvested in RIPA lysis buffer (Cwbiotech, Beijing, China) and Halt protease inhibitor cocktail (ThermoFisher, Rockford, USA) on ice. The primary antibodies used included the following: EYA4 (1:250 dilution; Santa Cruz, USA), E‐cadherin, vimentin, slug, MMP‐2, MMP‐13, Akt, p‐Akt Ser473, GSK‐3β, p‐GSK3β Ser9, Histone H2A.X, p‐Histone H2A.X Ser139, DNMT1, DNMT3A (1:1000 dilution; Cell Signaling Technology, USA) and β‐actin (1:5000 dilution; Abgent, Suzhou, China).
4
Investigating hnRNP E1 Knockdown in Cell Lines
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Human lung epithelial cell line A549 (ATCC), its hnRNP E1 knockdown (A549/E1KD or E1KD), normal mouse mammary epithelial (NMuMG) cell line, and its hnRNP E1 knockdown (NMuMG/E1KD or E1KD) derivative cells (Hussey et al, 2011 (link), 2012 (link)) were cultured in DMEM supplemented with 5% FBS and 5% FCS at 37°C in a 5% CO2 humidified chamber. Antibodies used in the work were against the following proteins: hnRNP E1 (mouse; Abnova; mouse, MBL; and rabbit, raised against purified hnRNP E1), TRF2 (rabbit, NB10056506; Novus), Hsp90 (mouse; SCBT), FLAG (rabbit; Sigma-Aldrich), 6XHis (rabbit; Cell signaling), phospho-ATM (MAB 2401; Abnova), ATM (2873S; Cell signaling), phospho-ATR, ATR (13934S; Cell signaling), phospho-p53 (9284S; Cell signaling), p53 (2524; Cell signaling), γ-H2A.X (rabbit, 9718S; Cell signaling), Histone H2A.X (7631T; Cell signaling), PCNA (mouse, Cat no. 2586S; SCBT), Usp1 (rabbit, 8033S; Cell signaling), Usp7 (mouse, HAUSP; SCBT), RPA32 (rat, 2208S; Cell signaling), Rad6 (R6A/R6B, rabbit, 4944S; Cell signaling), Rad18 (rabbit, 9040S; Cell signaling), and Fen1 (rabbit; SCBT). Plasmids containing hnRNP E1 and its three KH domains namely KH1, KH2, and KH3 have been described previously (Chaudhury et al, 2010b (link); Brown et al, 2016 (link)); each of the four ORFs contained an N-terminal GST tag.
5
Comprehensive Western Blotting for Protein Analysis
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Western blotting was performed as previously described46 (link). Electron transport proteins were detected using a total OXPHOS human western blot antibody cocktail (ab110411) from Abcam. The primary antibodies used are SFXN4 (Thermofischer (PA5-35980)), P-Histone H2A.X (Cell Signaling Technology (9718S)), Histone H2A.X (Cell Signaling Technology (2595S)), XPD (D3Z6I) (Cell Signaling Technology (11963)), NTH1 (Abcam (ab236912)), MUTYH (Abcam (ab228722)), FANCJ (BACH1/BRIP1) (Cell Signaling Technology (4578)), POLD1 (BD Biosciences (610972)), FTH46 (link), TFR1 (Thermo Fisher (13–6800)), HSP60 (Cell Signaling Technology (12165S)), AFG3L2 (Proteintech (14631-1-AP)), HSP90 (Cell Signaling Technology (4877), Tubulin (Cell Signaling Technology (12351)), β-Actin (Sigma Aldrich (A3854) C/EBPβ Antibody (Cell Signaling Technology #9008),Id2 (D39E8) (Cell Signaling Technology #3431) and RPA32/RPA2 Antibody (Cell Signaling Technology #52448).
6
Histone H2AX Phosphorylation Assay
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Primary antibodies included γH2AX (1:1000, #9718) and Histone H2AX (1:1000, #7631, Cell Signaling). The performance was based on the previous description [45 (link)].
7
Exosomal Marker and Content Evaluation
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Expression of exosomal markers and content were evaluated via dot blot analysis. Briefly, 5 μg of EV protein or cell lysates were added to the reducing buffer in a 1:1 ratio and blotted on nitrocellulose membrane. The membrane was blocked for nonspecific binding and was incubated with the primary antibodies of CD63 (1: 500) (Santa Cruz Biotechnology, Dallas, Texas), cleaved caspase-1 (1: 500) (Fisher Scientific, Grand Island, NY, USA), Histone (H2A.x) (1: 500) (Cell Signaling Technology Inc., Danvers, MA, USA), toll like receptor (TLR) 7 (1: 500) (Abnova, Neihu District, Taipei City, Taiwan), Ras binding Protein (Rab) 35 (1: 500) (BioFisher Scientific, Rockford, IL, USA), tumor susceptibility gene (TSG)101 (1: 500) (Fisher Scientific, Grand Island, NY, USA), and ALIX (1: 500) (Fisher Scientific, Grand Island, NY, USA), overnight at 4°C. Proteins were further detected using Horseradish peroxidase-(HRP-) conjugated secondary antibodies (goat anti-rabbit (1: 500-1: 1,000) (Novus Biologicals LLC, Centennial, CO, USA) or goat anti-mouse (1: 500-1: 1,000) (Fisher Scientific, Grand Island, NY, USA)). Targeted protein signals were detected using Super-Signal West Femto Maximum Sensitivity Substrate (Invitrogen, MA, USA), and images were developed using Bio-Rad ChemiDoc™ XRS+ System (Bio-Rad Laboratories, Hercules, CA, USA) (Ipinmoroti et al., 2021 (link)).
8
Western Blot Analysis of Protein Expression
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Proteins were extracted from cell lysates using standard methods. Proteins were separated by 10% or 12.5% running gel and stack gel electrophoresis, made of 40% acrylamide/bis solution, SDS, Tris and other components. Proteins were then transferred to the nitrocellulose membranes (Bio-rad, Hercules, California, USA), followed by blocking in 5% Bovine Fraction V (BSA, 9048-46-8, RPI (Research Products International), Mount Prospect, Illinois, USA) for 3h. After hybridization with primary antibodies at 4°C overnight, the membranes were washed and immunoblotted with secondary antibodies. Images were obtained by the Bio-Rad ChemiDoc XRS + System. Primary antibodies were as follows: PRDX2 (46855S, Cell Signaling Technology, Danvers, MA, USA), phospho–NF–κB-p65 (Ser536) (3033S, Cell Signaling Technology), NF-κB-p65 (D14E12) (8242s, Cell Signaling Technology), Phospho-Histone H2A.X (Ser139) (2577s, Cell Signaling Technology), Histone H2A.X (2595s, Cell Signaling Technology), Cleaved PARP (As214) (5625s, Cell Signaling Technology), PARP (46D11) (9532s, Cell Signaling Technology) and beta-Actin (A5441, Sigma-Aldrich, St. Louis, Missouri, USA).
9
Comprehensive Antibody Inventory for Cell Signaling
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Primary antibodies were mainly from Cell Signaling (Danvers, MA), including Phospho-MEK1/2 (1:1000, #9121), Phospho-p44/42 MAPK (1:1000, #9106), Phospho-Rb (Ser807/811) (1:1000, #9308), p21 (1:1000, #2946), p27 (1:1000, #2552), Cyclin D1 (1:1000, #2978), CDK4 (1:1000, #12790), CDK6 (1:1000, #3136), γH2AX (1:1000, #9718), Histone H2AX (1:1000, #7631), pSmad2/3 (1:1000, #8828), Smad2/3 (1:1000, #3102), and GAPDH (1:1000, #2118). Some other antibodies are pATM (1:1000, ab81292), ATM (1:1000, ab32420), E-cadherin (1:500, AAS89512C, Antibody Verify), N-cadherin (1:1000, 33–3900, ZYMED), Vimentin (1:500, AAS26482C, Antibody Verify). And the rest antibodies have been reported earlier [41 (link)].
10
Protein Expression Analysis by Western Blot
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Total protein was extracted using cell lysis solutions (Thermo Fisher Scientific, USA). Nucleoprotein was extracted using NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific™ Pierce™). Protein concentration was measured by BCA method. Then, 10% SDS-PAGE gel electrophoresis was performed using 20 μg of protein from each sample, followed by transfer from transmembrane to PVDF membrane (Bio-Rad, Hercules, CA, USA) at 25V for 1 h. Blocking was performed using 5% skimmed milk at room temperature for 1 h. After being washed with TBST, membranes were incubated with primary antibodies of IL-6 (Cell Signaling Technology, USA), NF-κB p65 (Cell Signaling Technology, USA), Histone H2A.X (Cell Signaling Technology, USA), E-cadherin (Santa Cruz Biotechnology, USA), α-SMA (Santa Cruz Biotechnology, USA), and β-actin (Sigma-Aldrich, USA) overnight at 4°C. After washing with TBST, membranes were further incubated with corresponding horseradish peroxidase-labeled IgG secondary antibody (1: 1000, Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.) at room temperature for 1 h. After washing with TBST, signals were detected using ECL (Sigma-Aldrich, USA) method.
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