Immunoblotting was performed using anti-phospho-RB (Cell Signaling, Danvers, MA, USA), anti-RB (GeneTex, Irvine, CA, USA), anti-phospho-Akt(Ser473) (Cell Signaling), anti-Akt (Cell Signaling), anti-phospho-Erk1/2(Thr202/Tyr204) (Cell Signaling), anti-Erk1/2 (Cell Signaling), anti-TOP2A (Cell Signaling), anti-CCND1 (Cell Signaling), anti-γH2AX (Cell Signaling), anti-CDK2 (GeneTex), anti-RAD51 (GeneTex), anti-CDK4 (Cell Signaling), and anti-GAPDH (GeneTex) antibodies followed by horseradish peroxidase-conjugated secondary antibodies and visualization using an enhanced chemiluminescence detection system. Immunohistochemical (IHC) sample preparation and staining with anti-GFAP (Genetex), anti-phospho-RB (Cell Signaling), anti-Ki-67 (Cell Signaling), and anti-γH2AX (Cell Signaling) antibodies were carried out as previously described [48 (link)]. Uncropped Western blot images are provided in Figure S5 .
Anti γh2ax
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom, China
Anti-γH2AX is a research-use antibody that recognizes the phosphorylated form of the histone variant H2AX, which is a marker for DNA double-strand breaks. This antibody can be used to detect and quantify the presence of DNA damage in cells.
Automatically generated - may contain errors
Lab products found in correlation
Anti cleaved caspase 3, by Cell Signaling Technology (13 mentions)
Anti p53, by Santa Cruz Biotechnology (11 mentions)
Anti β actin, by Cell Signaling Technology (11 mentions)
Anti caspase 3, by Cell Signaling Technology (10 mentions)
Anti bcl 2, by Cell Signaling Technology (9 mentions)
Anti p21, by Cell Signaling Technology (9 mentions)
Anti akt, by Cell Signaling Technology (8 mentions)
Anti rad51, by Abcam (7 mentions)
Anti chk2, by Cell Signaling Technology (7 mentions)
Anti parp, by Cell Signaling Technology (7 mentions)
Anti gapdh, by Santa Cruz Biotechnology (7 mentions)
Anti flag, by Merck Group (7 mentions)
Anti rad51, by Cell Signaling Technology (7 mentions)
Anti p chk2, by Cell Signaling Technology (7 mentions)
Anti β actin, by Merck Group (6 mentions)
Anti parp 1, by Santa Cruz Biotechnology (6 mentions)
Anti p21, by Santa Cruz Biotechnology (6 mentions)
Anti h2ax, by Cell Signaling Technology (6 mentions)
Anti p53, by Cell Signaling Technology (6 mentions)
Anti p p53, by Cell Signaling Technology (6 mentions)
180 protocols using anti γh2ax
1
Immunoblotting and Immunohistochemical Analysis
2
DNA Damage Response Pathway Analysis
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The following specific antibodies were used in this study: Anti‐γH2AX (#2577), anti‐γ‐H2AX (#9718) and anti‐GAPDH (#51332), anti‐H4K8ac (#2594), anti‐ATM (#2873), anti‐ATM (#92356), anti‐pATM (#5883S), anti‐ATR (#13934), anti‐pATR (#2853), anti‐CHK1(#2360), anti‐pCHK1 (#2348), anti‐CHK2 (#6334), anti‐pCHK2 (#2197), anti‐FLAG (8146), anti‐MYC (71D10) and anti‐TIP60 (#12058) were all purchased from Cell Signaling Technology (Boston, MA). anti‐TIP60 (#10398) was purchased from Abnova. anti‐ATM (#ET‐1606‐20) was purchased from Human Technology. Anti‐FAM135B (SAB2104963) was purchased from Sigma‐Aldrich (Merck Darmstadt, Germany). Anti‐FLAG (ab205606) and anti‐53BP1 (ab36823) antibodies were acquired from Abcam (England); anti‐GST (HT601) was acquired from Santa Cruz (Dallas, USA); bleomycin (BLM, radiomimetic drug), etoposide (ETO) and cisplatin (CDDP, a chemotherapy drug that can cause DNA double‐chain rupture as a DNA damage inducer) purchased from Selleck (America). We describe all the antibodies and drugs in Tables S1 and S2 .
3
Quantification of DNA Damage and Repair
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For γH2AX foci detection, cells were fixed with 4% paraformaldehyde and subsequently with methanol for 10 min at −20 °C. After permeabilization and blocking, the slides were incubated with anti-γ-H2AX (Cell Signalling Technology Danvers, MA, USA) and Alexa594-conjugated secondary antibody with 4′,6-diamidino-2-phenylindole (DAPI) staining. For immunoblot analysis, cells were lysed with Laemmli’s buffer and then sonicated, and protein concentration was measured by a Protein Assay (Bio-Rad, Hercules, CA, USA). Proteins were then subjected to electrophoresis on an SDS-polyacrylamide gel followed by transfer to a Sequi-BlotTM PVDF membrane (Bio-Rad) as described elsewhere. Immunoblot analysis was carried out using the following primary antibodies: anti-γ-H2AX (1:1000; Millipore, Burlington, MA, USA), anti-PARP-1 (1:1000; Cell Signalling Technology), and anti-β-actin (1:50,000, Sigma-Aldrich, St. Louis, MI, USA). The secondary antibodies were horseradish peroxidase-linked immunoglobulin, and immune complexes were detected using an enhanced chemiluminescence reaction kit (Millipore).
4
Immunoblotting of Autophagy and Apoptosis Markers
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Immunoblotting was performed using anti-phospho-RB (Cell Signaling, Danvers, MA, USA), anti-p62 (GeneTex, Irvine, CA, USA), anti-LC3B (Novus biologicals, Littleton, CO, USA), anti-caspase3 (Novus biologicals), anti-γH2AX (Cell Signaling), anti-PIK3C3 (GeneTex) and anti-GAPDH (GeneTex) antibodies, followed by visualization using horseradish peroxidase-conjugated secondary antibodies and an enhanced chemiluminescence detection system.
5
Immunofluorescence Staining for DNA Damage
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Immunofluorescence staining was performed as previously described45 (link). Briefly, cells were attached to glass slides and maintained in culture medium. Cells were subjected to fixation with 4% formaldehyde (for the detection of γH2AX) or 70% ethanol (for the detection of cisplatin-DNA adducts), permeabilization with 0.2% Triton X-100, followed by blocking with 5% BSA. Subsequently, cells were incubated with anti-γH2AX (Cell Signaling Technology, CST, #2577, Massachusetts, USA) or anti-cisplatin modified DNA antibody (Abcam, ab103261, Massachusetts, USA) at 4 °C overnight. Lastly, cells were stained with fluorescence-conjugated secondary antibodies and DAPI solution (Sigma-Aldrich, Missouri, USA), and then photographed with Leica fluorescence microscope (Leica Microsystems, Wetzlar, Germany).
6
Quantitative Immunoblotting and Immunofluorescence
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Primary antibodies used were anti-γH2AX (#2577, Cell Signaling), anti-GAPDH (MAB374, Millipore), anti-CAD (PA5-19913, Thermo Fisher), anti-STING (#13647, Cell Signaling), anti-phospho-STING (#19781, Cell Signaling), anti-ICAD (#9732, Cell Signaling), anti-cGAS (#79978, Cell Signaling), anti-E-cadherin (#3195, Cell Signaling) and anti-α-tubulin(T9026, Sigma–Aldrich). Secondary antibodies were (Western blotting) anti-rabbit IgG(H + L)-HRP (A6667, Sigma-Aldrich), anti-mouse IgG(H + L)-HRP (115-035-166, Dianova), (immunofluorescence) anti-mouse IgG(H + L)-Cy5 (715-175-151, Dianova) and anti-rabbit IgG(H + L)-Alexa488 (711-545-152, Dianova).
7
Western Blotting Protocol for Protein Analysis
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Western blotting was performed as described previously [62 (link)]. The primary antibodies used in this study include anti-γH2A.X (1:1000, 2577S, Cell Signal Technology, Danvers, MA, USA), anti-p53 (1:500, sc-6243, Santa Cruz Biotechnology, Dallas, TX, USA), anti-p53ser15 (1:1000, 9286S, Cell Signal Technology, Danvers, MA, USA), anti-cleaved PARP1 (1:1000, 5625S, Cell Signal Technology, Danvers, MA, USA), anti-MCL1 (1:1000, 39224S, Cell Signal Technology, Danvers, MA, USA), anti-BCL2 (1:1000, 2872S, Cell Signal Technology, Danvers, MA, USA), anti-ACTIN (1:150,000, MAB1501, Millipore, Burlington, MA, USA). The secondary antibodies used were HRP-linked anti-rabbit IgG (1:4000, 7074S, Cell Signaling Technology, Danvers, MA, USA) and HRP-linked anti-mouse IgG (1:4000, 7076S, Cell Signaling Technology, Danvers, MA, USA).
8
HNSCC Cell Culture and Reagents
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Cal27 and SCC25 cells were obtained from American Type Culture Collection. OSC19 cells were kindly provided by Dr. Jeffrey Myers (The University of Texas MD Anderson Cancer Center, Houston, TX, USA). Cells were grown in Dulbecco’s Modified Eagle’s Medium/F12 supplemented with 5% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 °C and 5% CO2. As extended in vitro cell culture and clonal expansion can lead to the emergence of new genotypes and altered cellular phenotypes over time, all experiments were performed on early passage cells between passage number 3 and 10. Chemical reagents were obtained as follows: cisplatin (purity >99.9%), crystal violet, and sodium orthovanadate from Sigma-Aldrich (St. Louis, MO, USA); gefitinib (purity >99%) from AstraZeneca (Macclesfield, UK); recombinant human epidermal growth factor (rhEGF) from Cell Sciences (Canton, MA, USA); alamarBlue and trypan blue from Invitrogen (Carlsbad, CA, USA); anti-EGFR, anti-phosphorylated EGFR (pEGFR), anti-ERK1/2, anti-pERK1/2, anti-poly ADP ribose polymerase (PARP), anti-β-actin, and anti-γH2AX from Cell Signaling (Beverly, MA, USA); annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) from Millipore (Bedford, MA, USA); and dimethylsulfoxide (DMSO), dithiothreitol (DTT), FBS and methanol from Thermo Fisher Scientific (Waltham, MA, USA).
9
Immunofluorescence Staining for Ki67 and γ-H2AX
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Cells were fixed with 4% formaldehyde in PBS at room temperature (RT) for 10 min. After fixation, cells were treated with 0.1% Triton X-100 in PBS for 10 min at RT. After blocked with 10% BSA for 2 h, cells were incubated with the primary antibody at 4 °C overnight, followed by washing in PBS for three times and incubation at RT for 2 h with the corresponding secondary antibody. The following antibodies were used at the indicated dilutions: anti-Ki67 (Abcam), anti-γ-H2AX (Cell signaling technology).
10
Comprehensive Protein Extraction and Analysis
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For protein extraction, all cell lines were lysed in RIPA buffer or in a less stringent buffer (20 mM Tris HCl pH 8, 137 mM NaCl, 1% NP-40, 2 mM ethylenediaminetetraacetic acid (EDTA)) for the SRSF3 ISO2 (SRSF3-TR) detection. The homogenates were subjected to western blot analyses as reported (26 (link)). Antibodies used were anti-SLU7 (Novus, 1:1000), anti-PARP1 (Santa Cruz SC-7150; 1:1000, CA, USA), anti-Actin (Sigma A2066; 1:6000, MO, USA), anti-Sororin (kindly provided by Dr JM Peters, Austria), anti-SRSF3 (Thermo Fisher 33-4200; 1:500 and MBL RN080PW; 1:500, MA, USA), anti-WAPL (Cell signaling 77428S; 1:1000, MA, USA), anti-H3S10P (Cell signaling #9701; 1:1000), anti-γ-H2AX (Cell signaling #2577S; 1:1000), anti-SRSF1 (Thermo Fisher 32-4600; 1:1000), anti-MAD2 (Bethyl laboratories a300-301A-M; 1:1000, TX, USA), anti-P21 (Santa Cruz SC-397; 1:1000), anti-RNaseH1 (Thermo Fisher PA5-30974; 1:1000) and anti-V5 (Invitrogene R960-25; 1:1000).
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