MIA PaCa-2, PANC-1, and PDX339 PDAC cells were treated with FFX (0.25 μM oxaliplatin, 0.5 μM irinotecan, and 1.25 μM 5-FU) for 24 h followed by a 30-min treatment with P-AscH− (5 mM). Protein was isolated using PhosphoSafe Extraction Reagent (EMD Millipore Corp, Burlington, Mass) as per manufacturer’s instructions. Protein (40 μg) was electrophoresed in a 4% to 20% Bio-Rad ready gel (Hercules, Calif) and then transferred to a Nitrocellulose membrane (Bio-Rad) at 60 V for 1 h. Membranes were blocked in 5% bovine serum albumin (RPI, Mount Prospect, Ill) for 1 h and then incubated with anti-γH2AX (phospho S139) antibody (1:1000, Abcam, Branford, Conn) or Beta-Tubulin (1:500, Developmental Studies Hybridoma Bank at the University of Iowa, #E7, Iowa City, Iowa) overnight at 4°C. Horseradish peroxidase (HRP)-conjugated goat anti-mouse (1:20,000, EMD Millipore Corp) was used as a secondary antibody. Blots were treated with SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, Mass) and exposed to autoradiography film.
Anti γh2ax phospho s139 antibody
Manufactured by Abcam
Sourced in China
Anti-γH2AX (phospho S139) antibody is a laboratory research tool used to detect the phosphorylation of histone H2AX at serine 139, a marker of DNA double-strand breaks. This antibody can be used in various applications such as Western blotting, immunohistochemistry, and flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Phosphosafe extraction reagent, by Merck Group (1 mentions)
Prolong gold, by Thermo Fisher Scientific (1 mentions)
Apotome module, by Zeiss (1 mentions)
Hoechst, by Merck Group (1 mentions)
Axiovert 200 m microscopy system, by Zeiss (1 mentions)
Tween 20, by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Irinotecan, by MedChemExpress (1 mentions)
Oxaliplatin, by MedChemExpress (1 mentions)
Gibco dmem medium, by Thermo Fisher Scientific (1 mentions)
Gibco rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Phospho sting ser365 d8f4w rabbit mab, by Cell Signaling Technology (1 mentions)
Sting d2p2f rabbit mab, by Cell Signaling Technology (1 mentions)
3 protocols using anti γh2ax phospho s139 antibody
1
Evaluating DNA Damage Response in PDAC
2
Quantification of DNA Damage Foci
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Cells were seeded in culture medium on glass coverslips at a density of 5×104cells/well in 12 well plates. 24h later, drugs alone or in combination were added for additional 24h. Cells were washed twice with PBS and fixed with 4% paraformaldehyde in PBS, 15min at room temperature. Cells were permeabilized with 0.05% Triton X-100 (Merck) /0.05% Tween-20 (Sigma-Aldrich) in PBS (5min) and incubated with anti-γH2A.X (phospho S139) antibody at 1μg/ml (Abcam) in PBS/1% BSA for 1h. Cells were washed twice with PBS and incubated with a DyLighttm 633 conjugated secondary antibody (Thermo Scientific) for 1h. After an additional PBS wash, cell nuclei were stained with 1μg/ml Hoechst (Sigma-Aldrich) (5min). Coverslips were mounted in ProLong® Gold (Molecular Probes) and fluorescence was visualized using the Axiovert200M microscopy system (Zeiss, Le Pecq, France) with ApoTome module (X63 and numerial aperture 1.4).
3
STING Signaling Pathway Assessment
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UNC2250 was purchased from Adooq Bioscience (Nanjing, China). Oxaliplatin and irinotecan were purchased from MedChemexpress (Wuhan, China). Cytometric Beads for cytokine detection were purchased from BD Bioscience (Shanghai, China). BD Biosciences (Shanghai, China). DiD was purchased from Beyotime Biotechnology (Shanghai, China). Granulocyte/macrophage-colony stimulating factor (GM-CSF) was purchased from Dakewe (Shenzhen, China). Macrophage-colony stimulating factor (M-CSF) was purchased from Abcolonal (Wuhan, China), NGS™ dsDNA HS Assay Kit was purchased from Wuhan ABP-Biosciences Co., Ltd. (Wuhan, China). Gibco DMEM medium and Gibco RPMI 1640 medium were purchased from Thermo Fisher Scientific Co., Ltd. (Shanghai, China). Anti-γ H2A.X (phospho S139) antibody was purchased from Abcam (Shanghai, China). STING (D2P2F) Rabbit mAb was purchased from Cell Signaling Technology (Shanghai, China). Phospho-STING (Ser365) (D8F4W) Rabbit mAb was purchased from Cell Signaling Technology (Shanghai, China).
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