Axolotl AL1 fibroblast cells were kept at room temperature with modified L-15 media (0.7x, 5% FBS, 2 mM L-Glutamine, 1x Insulin-Transferrin-Selenium, 1x Penicillin-Streptomycin). AL1 cells were plated on glass coverslips and exposed to UV under a G30T8 UV lamp for 15 min. Controls were covered with aluminum foil. Cells were left to recover for 4 hr before fixation with 4% paraformaldehyde for 15mins before blocking and antibody incubations. Antibodies used were: phospho-histone H2A.X (Ser139/Tyr142) (#5438, Cell Signaling Technology), anti-gamma H2A.X (phospho S139) (#ab11174, Abcam), and anti-gamma H2A.X (phospho S139) (#ab81299, Abcam). Images were captured using a Nicon Eclipse Ni fluorescent microscope equipped with a Nikon DS-Ri2 camera.
Anti gamma h2a x phospho s139
Manufactured by Abcam
Sourced in United Kingdom
Anti-gamma H2A.X (phospho S139) is a laboratory research antibody that detects the phosphorylation of serine 139 on the histone variant H2A.X. This post-translational modification is a marker of DNA double-strand breaks and is involved in the cellular DNA damage response.
Automatically generated - may contain errors
Lab products found in correlation
Ds ri2 camera, by Nikon (1 mentions)
Eclipse ni fluorescent microscope, by Nikon (1 mentions)
Phospho histone h2a x ser139 tyr142, by Cell Signaling Technology (1 mentions)
Fluorescence microscope, by Leica (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Imaging chamber, by Ibidi (1 mentions)
Donkey anti goat igg h l alexa fluor 488 conjugate, by Thermo Fisher Scientific (1 mentions)
Anti gfp, by Abcam (1 mentions)
Lsm 700 confocal microscope, by Zeiss (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Anti egfr, by Abcam (1 mentions)
Anti egfr, by Proteintech (1 mentions)
Anti golph3, by Abcam (1 mentions)
4 protocols using anti gamma h2a x phospho s139
1
Quantifying DNA Damage in Axolotl Fibroblasts
2
Immunofluorescence Staining of DNA Damage and Telomere Proteins
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The cells from various groups were fixed in 4% formaldehyde at room temperature (R.T) for 10 min and permeabilized in 0.5% Triton X-100 in PBS at R.T for 20 min. After washing with PBS, the cells were blocked with 5% bovine serum albumin (BSA) in PBS at R.T for 1 h and thereafter incubated with primary antibody diluted 1:100 at 4°C overnight. Then, after washing with PBS again, the cells were reacted with according secondary antibody (Abcam, Cambridge, UK) at R.T for 1 h. The cells were restained with 4′-6-diamidino-2-phenylindole (DAPI) (Invitrogen, Carlsbad, CA, USA). Fluorescent imaging was conducted with a fluorescence microscope (Leica, Wetzlar, Germany). Mouse monoclonal anti-gamma H2A.X (phospho S139) were obtained from Abcam (Abcam, Cambridge, UK). Rabbit polyclonal anti-TRF2 antibody was purchased from Santa Cruz (Santa Cruz Biotechnology, Santa Cruz, CA, USA).
3
DNA Damage Quantification in Transfected Cells
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293T cells were transfected with pLVX-overexpression or vector control and seeded onto imaging chambers (Ibidi Cat #: 81156) pre-coated with poly-D-lysine. At about 70 h post-transfection, cells were fixed with 3.7% formaldehyde in DPBS for 15 min at room temperature, washed twice with DPBS, permeabilized and blocked in DPBS supplemented with 0.5% Triton X and 1% BSA for 15 min at room temperature, and incubated overnight at 4 degC with primary antibodies anti-gamma H2A.X (phospho S139) (Abcam, Cat #: ab2893, 1:1000), and anti-GFP (Abcam, Cat #: 6673, 1:500). On the following day, the imaging chambers are washed thrice with DPBS, and incubated with secondary antibodies donkey anti-Goat IgG (H + L) Alexa Fluor® 488 conjugate (Thermofisher, Cat #: A11055, 1:500) and donkey anti-Rabbit IgG (H + L) Alexa Fluor® 594 conjugate (Thermofisher, Cat #: A21207, 1:250), and DAPI (1 μg/mL) for 1.5 h at room temperature. The chambers were washed thrice with DPBS before imaging using Zeiss LSM700 confocal microscope.
4
Protein Extraction and Western Blot Analysis
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Cytoplasmic and nuclear extracts were acquired using the NE-PER ® nuclear and cytoplasmic extraction kit (Pierce, Rockford, IL, USA). Whole-cell protein was extracted with RIPA lysis buffer (Beyotime Biotechnology) and the protein concentration was determined with a BCA protein assay kit (Beyotime Biotechnology). Briefly, proteins were separated by 6-15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes (Merck Millipore, Darmstadt, Germany).
Membranes were blocked in 5% skim milk (BD/Difco, Sparks, MD, USA) for 1 h, and then incubated with different primary antibodies at 4°C overnight. The primary antibodies utilized were: anti-GOLPH3 (1:1000; Abcam), anti-EGFR (1:1000; Protein Tech Group), anti-gamma H2AX (phospho S139) (1:1000; Abcam), anti-ubiquitin For co-immunoprecipitation (co-IP), 2 g of anti-DNA-PK, anti-EGFR or control IgG antibody was incubated with 4 mg of cell lysate, followed by capturing with protein-A/G agarose. The beads were then washed extensively and suspended in SDS loading buffer for western blot analysis.
Membranes were blocked in 5% skim milk (BD/Difco, Sparks, MD, USA) for 1 h, and then incubated with different primary antibodies at 4°C overnight. The primary antibodies utilized were: anti-GOLPH3 (1:1000; Abcam), anti-EGFR (1:1000; Protein Tech Group), anti-gamma H2AX (phospho S139) (1:1000; Abcam), anti-ubiquitin For co-immunoprecipitation (co-IP), 2 g of anti-DNA-PK, anti-EGFR or control IgG antibody was incubated with 4 mg of cell lysate, followed by capturing with protein-A/G agarose. The beads were then washed extensively and suspended in SDS loading buffer for western blot analysis.
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