Pine needle oil [dissolved in dimethyl sulfoxide (DMSO), ≤0.1%], RNase and propidium iodide (PI) solution were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Anti-γ-H2AX (cat no. 05636; 1:1,000) antibody was purchased from EMD Millipore Billerica, MA, USA), ATM (cat no. 2873; 1:500), p-ATM (cat no. 13050; 1:1,000), p-p53 (S15; cat no. 9286; 1:1,000), p-CDC25C (S216; cat no. 4901; 1:500) p-CHK2 (T68; cat no. 2661; 1:1,000) and CHK2 (cat no. 2662; 1:1,000) antibodies were purchased from Cell Signaling Technology, Inc., (Danvers, MA, USA). CDC25C (cat no. sc-327; 1:1,000), β-actin (cat no. sc-47778; 1:1,000), anti-H2AX (cat no. sc-54606; 1:200), p53 (cat no. sc-98; 1:500) antibodies, goat anti-rabbit (cat no. sc-2030; 1:3,000) and anti-mouse secondary (cat no. sc-2031; 1:3,000) antibodies were purchased from Santa Cruz Biotechnology, Inc., (Dallas, TX, USA). Protein extraction solution kit was purchased from Beijing SBS Genetech Co., Ltd., (Beijing, China). Dulbecco's modified Eagle's medium and bovine serum albumin were purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The HepG2 cell line was purchased from the China Center for Type Culture Collection of Wuhan University (Wuhan, China). This cell line was originally thought to be a hepatocellular carcinoma, but is now known to be a hepatoblastoma cell line (21 (link)).
Anti h2ax
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Anti-H2AX is a laboratory product used for the detection and quantification of phosphorylated H2AX, a histone variant that is a marker of DNA double-strand breaks. It can be used in various applications, such as cell-based assays and immunohistochemistry, to study DNA damage and repair processes.
Automatically generated - may contain errors
Lab products found in correlation
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Propidium iodide solution, by Merck Group (1 mentions)
Anti γh2ax, by Merck Group (1 mentions)
Cdc25c, by Santa Cruz Biotechnology (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
P cdc25c, by Cell Signaling Technology (1 mentions)
P chk2, by Cell Signaling Technology (1 mentions)
P atm, by Cell Signaling Technology (1 mentions)
Anti chk2, by Cell Signaling Technology (1 mentions)
Ecl western blotting kit, by Cytiva (1 mentions)
Amersham hybond ecl nitrocellulose membranes, by Cytiva (1 mentions)
Hybond, by Cytiva (1 mentions)
Alexa fluor 488 donkey anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Scramble sirna, by Santa Cruz Biotechnology (1 mentions)
Anti bcl 2, by Merck Group (1 mentions)
N6 isopentenyladenosine ipa, by Merck Group (1 mentions)
Anti nf κb p65 d14e12, by Cell Signaling Technology (1 mentions)
Anti p38, by Merck Group (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
5 protocols using anti h2ax
1
Investigating DNA Damage Responses in HepG2 Cells
2
Western Blot Analysis of DNA Damage Markers
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Western blot analysis was performed according to our established protocol [39 (link), 54 (link), 83 (link)–85 (link)]. Briefly, 50 µg of total cell lysate protein was separated on a SDS-PAGE gel and transferred onto Amersham Hybond ECL nitrocellulose membranes (Amersham, Baie d’Urfe, QC). Blots were treated with 5% skim milk and incubated at 4°C overnight with the following antibodies: anti-H2AX (1:1000, Santa Cruz), anti-γH2AX (1:1000, Upstate), anti-phosph-CHK2 (T68) (1:500, Cell Signaling), and anti-CHK2 (1:1000, Cell Signaling). The blots were then incubated with the specific HRP-conjugated secondary antibodies at room temperature for one hour, followed by developing signals using an ECL Western Blotting Kit (Amersham, Baie d’Urfe, QC).
3
Investigating DNA Damage Response Signaling
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N6-isopentenyladenosine (iPA) (Sigma-Aldrich, St. Louis, MO) was dissolved in DMSO and added to cell cultures at the indicated concentration. For Western blot analysis the following antibodies were used: anti-RAD51, anti-pCHK1 (S345), rabbit anti-pCHK2 (T68), anti-p-ATM (S1981), anti-p-BRCA1 (S1524), anti-p-ATR (S428), anti-p-AKT (S473), anti-PARP, anti- p-JAK2 (Tyr 1007/1008), anti-JAK2, anti-NF-κB p65 (D14E12), and anti-Caspase-3 were purchased from Cell Signaling Technology (Danvers, MA), anti-CHK2, anti-STAT5 a/b, anti-H2AX, anti-γ-H2AX (Ser139), anti-β-actin, anti-BRCA1, anti-p-STAT5a/b (Tyr 694/699), anti-p-p38 (Tyr182) were purchased from Santa Cruz Biotechnology (Dallas, TX), anti-CHK1 from Abcam (Cambridge, UK), anti-BCL-2 and anti-p38 from Sigma-Aldrich Inc. (St Luis, MO). For fluorescence microscopy anti-RAD51 (Cell Signaling Technology, Danvers, MA), anti-γ-H2AX (Santa Cruz Biotechnology Dallas, TX) and Alexa Fluor 488 donkey anti-rabbit IgG (Jackson ImmunoResearch, Cambridge, UK) and DyLight 594 goat anti-mouse IgG (Abcam, Cambridge, UK) were used. STAT5a/b-siRNA and scramble-siRNA were purchased from Santa Cruz Biotechnology (Dallas, TX).
4
Western Blot Analysis of Cell Lysates
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For whole cell lysates, cells were resuspended in 1X SDS sample buffer (25mM Tris–HCl pH 6.8, 1.5mM EDTA, 20% glycerol, 2% SDS, 5% b-mercaptoethanol, 0.0025% Bromophenol blue). For Western blot analysis, equal quantity of cell lysates were separated by SDS-polyacrylamide gel electrophoresis, transferred to PVDF membrane (VWR) and probed with the following primary antibodies: anti-NF-YA (Santa Cruz, sc-17753), anti-NF-YB (GeneSpin), anti-p53 (Santa Cruz, sc-126), anti-PARP1 (Santa Cruz, sc-8007), anti-H2AX (Santa Cruz, sc-101696), anti-p21 (Millipore, 05-345), anti-E2F1 (Bethyl, A300-766A), anti-cJun (Bethyl, A302-958A), anti-cMyc (Santa Cruz, sc-764), anti-Fos (Santa Cruz, sc-52), anti-p63 4A4 (Santa Cruz, sc-A0311), anti-actin (Santa Cruz, sc-1616), anti-tubulin (Sigma Aldrich, T-6074), anti-E6 (Santa Cruz, sc-365089). Chemiluminescent detection reagent was purchased from Millipore Spa (Luminata Classico and Forte Western HRP).
5
Immunofluorescent Staining for Astrocytes and DNA Damage
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For intra-cellular markers, staining was performed on living cells followed by fixation and permeabilization. Anti-Glial fibrillary acidic protein (GFAP; Cat# Z0334, RRID: AB_10013382, Agilent, Santa Clara, CA, USA, 1:50) was used to identify astrocytes, anti-H2AX (Cat# sc-517348, RRID:AB_2783871, Santa Cruz biotechnology Inc., Dallas, TX, USA, 1:100) was used for evaluation of DNA damage response. Goat anti-rabbit Alexa Fluor 488 (Cat# A-11034, RRID: AB_2576217, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA, 1:200) and goat anti-mouse Alexa Fluor 555 (Cat# A28180, RRID:AB_2536164, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA, 1:200) were used as secondary antibodies appropriately. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Cat# H-1200, RRID:AB_2336790, Vector Laboratories, Burlingame, CA, USA). Quantification was performed using ImageJ software (NIH, public domain software) by measuring positively stained cells relative to total DAPI. Quantifications are represented as mean percentages from total DAPI+ cells ± SD and are from at least 15 random fields captured in three or more independent experiments.
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