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Epr9514

Manufactured by Abcam
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EPR9514 is an anti-Phospho-Histone H2A.X (Ser139) antibody. It is a recombinant rabbit monoclonal antibody that recognizes histone H2A.X phosphorylated at serine 139. This antibody can be used in various applications such as Western blot, immunohistochemistry, and immunocytochemistry.

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Lab products found in correlation

Lionheart microscope, by Agilent Technologies (1 mentions) Bs 0061r, by Bioss Antibodies (1 mentions) Ab209845, by Abcam (1 mentions) Bs 0296g, by Bioss Antibodies (1 mentions) Af6188, by Affinity Biosciences (1 mentions) Af3189, by Affinity Biosciences (1 mentions) Bs 2188r, by Bioss Antibodies (1 mentions) Bs 0295g, by Bioss Antibodies (1 mentions) Bs 0032r, by Bioss Antibodies (1 mentions) Bs 10442r, by Bioss Antibodies (1 mentions) Af0120, by Affinity Biosciences (1 mentions) Ripa lysis buffer, by Beyotime (1 mentions) Bca protein assay, by Beyotime (1 mentions)

3 protocols using epr9514

1

Quantifying Necroptosis Markers in Tissue

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Biopsies were dissected as indicated in each experiment and infused with 4% neutral-buffered formaldehyde, fixed for 48 h, dehydrated in a graded ethanol series and xylene, and finally embedded in paraffin. Paraffin sections (3–5 mm) were stained with periodic acid–Schiff reagent, according to the standard routine protocol. Immunohistochemistry for S358 pMLKL monoclonal antibody (Abcam, EPR9514) was performed according to routine protocols with dilutions of 1:500 to 1:1000. Micrographs were digitalized using an AxioCam MRm Rev. 3 FireWire camera and AxioVision Rel. 4.5 software (Zeiss). The percentage of the number of pMLKL+ cells to the total number of the endothelial cells was quantified by an experienced pathologist in a double-blind manner (that is, all slides were randomized and coded, independently of individual patient sample or treatment).
Gong Y.N., Guy C., Olauson H., Becker J.U., Yang M., Fitzgerald P., Linkermann A, & Green D.R. (2017). ESCRT-III acts downstream of MLKL to regulate necroptotic cell death and its consequences. Cell, 169(2), 286-300.e16.
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2

Immunofluorescence Staining of pMLKL and IL-8

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Frozen sections (5 μm) were fixed with 4% paraformaldehyde-PBS 20 min at room temperature, permeabilized with 0.3% Triton X-100 in 1X PBS, blocked in 3% BSA (or 10% FBS) in 1X PBS, stained with primary antibodies for overnight at 4°C, followed by staining with 2° antibodies (1 hr) and DAPI stain. Samples were covered with coverglasses and imaged using Lionheart microscope (Biotek). S358 pMLKL monoclonal antibody (Abcam, EPR9514, 1:500) and human IL-8 (CXCL8) Monoclonal Antibody (3IL8-H10, Thermo Fisher, 1:1000) were used to perform the immunofluorescence staining. Samples were imaged using Lionheart microscope (Biotek).
Wang W., Prokopec J.S., Zhang Y., Sukhoplyasova M., Shinglot H., Wang M.T., Linkermann A., Stewart-Ornstein J, & Gong Y.N. (2022). Sensing plasma membrane pore formation induces chemokine production in survivors of regulated necrosis. Developmental cell, 57(2), 228-245.e6.
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3

Western Blot Analysis of Necroptosis Signaling

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Tissues and cells were lysed using RIPA lysis buffer (Beyotime Biotechnology) containing protease and phosphatase inhibitor, homogenated, and centrifuged. The protein concentration was determined by BCA protein assay (Beyotime Biotechnology). Equal concentrations of proteins were separated in a 10% SDS-PAGE and transferred to PVDF membrane. The membrane was blocked using 5% milk in TBST, probed with primary antibody, RIPK1 (17519-1-AP, Proteintech, 1:1000), MLKL (GTX107538-100, GeneTex, 1:1000), RIPK3 (17563-1-AP, Proteintech, 1:1000), p-MLKL (EPR9514, Abcam, 1:1000), PKA (bs-0520R, Bioss, 1:1000), p-PKA (#9621, Cell signaling, 1:1000), CREB (AF6188, Affinity, 1:1000), p-CREB (AF3189, Affinity, 1:1000), P2Y14R (bs-12028R, Bioss, 1:1000), GSDMD (ab209845, Abcam, 1:1000), Caspase1 p20 (bs-10442R, Bioss, 1:1000), Bcl-2 (bs-0032R, Bioss, 1:1000), Bax (AF0120, Affinity, 1:1000), Caspase-3 p-17 (Cell Signaling Technology, #9664, 1:1000), β-actin (bs-0061R, Bioss, 1:1000), GAPDH (BS-2188R, Bioss, 1:1000) and corresponding HRP-conjugated antibodies Goat Anti-Rabbit IgG H&L Antibody (bioss, Bs-0295G, 1:8000) or Goat Anti-Mouse IgG H&L Antibody (bioss, Bs-0296G, 1:8000). Densitometry was quantified using ImageJ software.
Liu C., Wang H., Han L., Zhu Y., Ni S., Zhi J., Yang X., Zhi J., Sheng T., Li H, & Hu Q. (2024). Targeting P2Y14R protects against necroptosis of intestinal epithelial cells through PKA/CREB/RIPK1 axis in ulcerative colitis. Nature Communications, 15, 2083.
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