Catalog numbers and dilution fold are listed in parentheses. The following antibodies were purchased from Cell Signaling Technology: Anti-Bak (3792; 1:1000), anti-Bax (2772; 1:1000), anti-Bcl-2 (2870; 1:1000), anti-Bcl-xL (2764; 1:500), anti-CHK1 (2360; 1:1000), anti-phospho-CHK1 (S345; 2348; 1:1000), anti-cleaved caspase-3 (9661; 1:1000), anti-cleaved caspase-7 (9491; 1:1000), anti-cleaved caspase-8 (9496; 1:500), anti-cleaved caspase-9 (9501; 1:1000), anti-cytochrome c (4272; 1:500), anti-H2A.X (7631; 1:1000), anti-phospho-H2A.X (S139; 9718; 1:1000), anti-p53 (2524; 1:1000), anti-p53 (2527; 1:1000), anti-phospho-p53 (S6; 9285; 1:1000), anti-phospho-p53 (S15; 9284; 1:1000), anti-p21 (2946; 1:1000), and anti-voltage-dependent anion channel (VDAC; 4866; 1:1000). The following antibodies were purchased from Santa Cruz Biotechnology: Anti-CD95 (1023; 1:500), anti-cyclophilin 40, also known as anti-CYPD (137157; 1:400), anti-His tag (803; 1:500), anti-lamin A (20680; 1:1000), anti-MDM2 (965; 1:500), and anti-ubiquitin (8017; 1:200). Anti-PEPD (Ab86507; 1:500) and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; MAB374; 1:5000) were purchased from Abcam and EMD Millipore, respectively. Anti-mouse IgG-horseradish peroxidase (IgG-HRP; NA931V; 1:4000–5000) and anti-rabbit IgG-HRP (NA934V; 1:5000) were purchased from GE Healthcare.
Anti phospho h2ax
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-phospho-H2AX is a laboratory product that detects the phosphorylation of the histone variant H2AX. This phosphorylation event is a marker of DNA double-strand breaks.
Automatically generated - may contain errors
Lab products found in correlation
Anti h2ax, by Cell Signaling Technology (3 mentions)
Anti phospho p53, by Cell Signaling Technology (3 mentions)
Anti gapdh, by Merck Group (3 mentions)
Anti cleaved caspase 7, by Cell Signaling Technology (2 mentions)
Anti bax, by Cell Signaling Technology (2 mentions)
Anti bcl 2, by Cell Signaling Technology (2 mentions)
Anti p21, by Cell Signaling Technology (2 mentions)
Anti p21, by BD (2 mentions)
Anti parp, by Cell Signaling Technology (2 mentions)
Anti phospho atm, by Cell Signaling Technology (2 mentions)
Anti phospho chk2, by Cell Signaling Technology (2 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Apotome, by Zeiss (2 mentions)
Anti chk1, by Cell Signaling Technology (1 mentions)
Anti cleaved caspase 9, by Cell Signaling Technology (1 mentions)
Anti cleaved caspase 8, by Cell Signaling Technology (1 mentions)
Anti p53, by Cell Signaling Technology (1 mentions)
Anti phospho chk1, by Cell Signaling Technology (1 mentions)
Anti cd95, by Santa Cruz Biotechnology (1 mentions)
Anti cypd, by Santa Cruz Biotechnology (1 mentions)
18 protocols using anti phospho h2ax
1
Comprehensive Antibody Panel for Cell Signaling Analysis
2
Protein Extraction and Western Blot Analysis
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Extraction of proteins from mouse colonic mucosa and Western blot analysis were performed as previously described [40 (link),41 (link)]. The primary antibodies used were anti-LC3 (#L8918, Sigma-Aldrich, Saint-Louis, MO, USA), anti-phospho-H2AX (#2577, Cell Signaling) and anti-α-tubulin (#2144, Cell Signaling). The secondary antibody used was HRP-conjugated anti-rabbit (#7074, Cell Signaling). Blots were detected using the Enhanced Chemiluminescence Detection kit (RPN2108, Amersham Biosciences, Buckinghamshire, UK) and revealed using the ChemiDocTM XRS System (BioRad, Hercules, CA, USA).
3
Organoid Viability and Signaling Assay
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When organoids reached a density of 70–80% they were treated for 48 h with BaP concentrations that resulted in 30%, 50% and 80% viability. Organoids were then harvested and incubated in TryLE for 10 min at 37 °C to remove the membrane matrix. The organoid pellet was washed with cold PBS and lysed in 62.5 mM Tris (pH 6.8), 1 mM EDTA (pH 8.0), 2% sodium dodecyl sulfate, 10% glycerol, 1X HaltTM Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific, Waltham, MA, USA; #78442). Western blotting was carried out as described [48 (link)]. The primary antibodies used were: anti-p21 (1:2000; BD Bioscience, Franklin Lakes, NJ, USA; #BD556431), anti-phospho-H2AX (Ser139, 1:1000; Cell Signalling, Danvers, MA, USA; #9718S), anti-phospho-CHK2 (T68, 1:1000; Cell Signalling, #2197S), anti-phospho-p53 (Ser15, 1:2000; Cell Signalling, #9284S) and anti-GAPDH (1:25,000; Chemicon, Tenecula, CA, USA; #MAB374).
4
Western Blot Analysis of Cellular Stress Markers
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Protein extraction and western blot analysis were performed as previously described [52 (link)]. Anti-Beclin-1, anti-cleaved caspase 7, anti-CHOP, anti-GRP78, anti-phospho-H2AX, anti-LC3, anti-PARP, and anti-p62 were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-GRP94 was obtained from Enzo Life Sciences (Farmingdale, NY, USA). Anti-α-tubulin was obtained from Abcam (Eugene, OR, USA).
5
Immunohistochemical Analysis of DNA Damage
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The sections were deparaffined in Histoclear for 15 min (2 times), rehydrated in ethanol diminishing gradient (100%, 96%, 75% and water: 1 min each) and unmasked in Tris-EDTA buffer (10 mM Tris-Base, 1 mM EDTA, pH 9) during 20 min at 95 °C. The sections were incubated with blocking buffer (1% bovin serum albumin in PBS) for 1 h at room temperature and then with anti-phospho-H2AX (dilution 1/500, #9718, Cell Signaling, Danvers, MA, USA) overnight at 4 °C. After several washes with PBS, the sections were incubated with the corresponding secondary antibody coupled with peroxydase (dilution 1/500, #111-065-003, Jackson Immunoresearch, Ely, Cambridgeshire, United Kingdom) for 2 h at room temperature. Revelation was performed using 3,3′-Diaminobenzidine (#SK-4800, NovaRED, Vector Laboratories, Burlingame, CA, USA). The sections were counterstained with Mayer hematoxilin (Diapath, Martinengo (BG) Italy) for 10 sec, rinsed under running water, dehydrated and mounted in Eukitt mounting solution. The microscopic images were acquired using the Scanner Zeiss Axioscan Z1 (Zeiss, Jena, Germany) and analysed using ZEN 2 software.
6
Cell Signaling Pathway Profiling
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Whole cell lysates were extracted using radioimmunoprecipitation assay (RIPA) buffer together with a 1 × phosphatase inhibitor cocktail (PhosSTOP, Roche, Basel, Switzerland). The primary antibodies used were as follows: anti-CTPS2 (ab196016, Abcam, Cambridge, UK), anti-cyclin B1 (ab32053,Abcam), anti-CDK1 (ab18, Abcam), anti-phospho-H2AX (7631, Cell Signaling Technology, MA,USA), anti-phospho-ATM (13050, Cell Signaling Technology), anti-phospho-BRCA1 (14823, Cell Signaling Technology), anti-Bcl-2 (4223, Cell Signaling Technology), anti-Bax (14796, Cell Signaling Technology), anti-cleaved PARP (5625, Cell Signaling Technology), anti-p21 (2947, Cell Signaling Technology), β-actin (TA-08, Zhongshan Goldenbridge), and anti-GAPDH (TA-09, Zhongshan Goldenbridge, Beijing, China).
7
Analysis of DNA Damage Response
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Cells were lysed using cell lysis buffer (Cell Signaling) with complete protease inhibitor cocktail (1:1,000, Roche) and Halt phosphatase inhibitor cocktail (1:100, Thermo). After total protein quantification using a Bradford reagent (Bio-Rad), 20 μg of cell lysate protein was resolved by 4–15% SDS-PAGE (Bio-Rad) under denaturing conditions, and transferred to PVDF membranes. Membranes were blocked in 5% BSA and then incubated with anti- phospho-H2AX (1:1,000, Cell Signaling), phospho-Chk1-Ser317 (1:1,000; Cell Signaling), phospho-Chk2-Thr68 (1:1,000, Cell Signaling), caspase-3 (1:1,000; Cell Signaling), cleaved caspse-3 (1:1,000; Cell Signaling), PARP (1:1,000; Cell Signaling), and GAPDH (1:5,000, Cell Signaling) antibodies, followed by incubation with a secondary antibody conjugated with horseradish peroxidase (HRP) (1:2,000; Cell Signaling). Signals were visualized using ECL Prime Western Blotting Detection reagent (Amersham).
8
Western Blot Analysis of Cytoskeletal Proteins
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Cells were lysed in RIPA Buffer (Sigma-Aldrich) and protein concentration was determined by using the BCA assay kit (Sigma-Aldrich). Total protein (40 μg) was resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on a 10% or 5% gel and electrotransferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The membranes were then incubated in 5% nonfat dry milk in Tris-buffered saline (TBS, pH 7.6) and then overnight at 4 °C with a rabbit monoclonal anti-DHC2 (#ab122525; Abcam) at a dilution of 1:1000, anti-KIF2B (#ab98214; Abcam) at a dilution of 1:2000 or anti-Phospho-H2A.X (#9718; Cell Signaling Technology) at a dilution of 1:1000. Membranes were then incubated at 37 °C for 1 h with an HRP-conjugated anti-rabbit IgG antibody (#7074; Cell Signaling Technology) at a dilution of 1:2000. After three washes with TBS, membranes were briefly incubated with chemiluminescent HRP substrate (#WBKLS0100; Millipore) and were photo-developed in Image Station 2000 MM (Kodak, Rochester, Minnesota, USA). Quantity One 4.6.2 (Bio-Rad Laboratories, Hercules, CA, USA) was used to quantify the density of the target protein in the membranes.
9
Quantifying DNA Damage and Repair Foci
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For imaging γ-H2AX and Rad51
foci, the cells growing on the coverslips were treated as indicated.
Then the cells were fixed with 3.7% formaldehyde and permeabilized
with 0.5% triton X-100. The cells were washed with 1× PBS and
blocked with 3% BSA before incubating with primary antibodies. The
following primary antibodies were used: anti-phospho-H2AX (rabbit,
Cell Signaling Technology, catalogue no. 9718, 1:400); anti-Rad51
(rabbit, Santa Cruz Biotechnology, catalogue no. sc-8349, 1:100);
anti-LA (mouse, Sigma, catalogue no. SAB4200236, 1:1000); anti-FLAG
M2 (mouse, Sigma, catalogue no. F3165, 1:400). The cells were then
incubated with secondary antibodies for 1 h at room temperature. Cy-3-conjugated
donkey antirabbit secondary antibody (Jackson Immunoresearch) was
used at 1:1000 dilution. Coverslips were mounted in ProLong Gold AntiFade
reagent with DAPI (Life Technologies), and images were acquired with
a fluorescence microscope ApoTome (Zeiss). The cells were considered
foci-positive if >10 foci were observed per nucleus. DAPI was used
to count cell nuclei. Around 100 cell nuclei were analyzed for each
experiment, and results are shown as the average of three independent
cell preparations. For colocalization analysis, the Z-stack of images were reconstructed, and colocalization Pearson correlation
coefficients were determined using the Coloc module in the Imaris
software package (Bitplane).
foci, the cells growing on the coverslips were treated as indicated.
Then the cells were fixed with 3.7% formaldehyde and permeabilized
with 0.5% triton X-100. The cells were washed with 1× PBS and
blocked with 3% BSA before incubating with primary antibodies. The
following primary antibodies were used: anti-phospho-H2AX (rabbit,
Cell Signaling Technology, catalogue no. 9718, 1:400); anti-Rad51
(rabbit, Santa Cruz Biotechnology, catalogue no. sc-8349, 1:100);
anti-LA (mouse, Sigma, catalogue no. SAB4200236, 1:1000); anti-FLAG
M2 (mouse, Sigma, catalogue no. F3165, 1:400). The cells were then
incubated with secondary antibodies for 1 h at room temperature. Cy-3-conjugated
donkey antirabbit secondary antibody (Jackson Immunoresearch) was
used at 1:1000 dilution. Coverslips were mounted in ProLong Gold AntiFade
reagent with DAPI (Life Technologies), and images were acquired with
a fluorescence microscope ApoTome (Zeiss). The cells were considered
foci-positive if >10 foci were observed per nucleus. DAPI was used
to count cell nuclei. Around 100 cell nuclei were analyzed for each
experiment, and results are shown as the average of three independent
cell preparations. For colocalization analysis, the Z-stack of images were reconstructed, and colocalization Pearson correlation
coefficients were determined using the Coloc module in the Imaris
software package (Bitplane).
10
Antibody-Based DNA Damage Analysis
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