Ab124744

Manufactured by Abcam

Sourced in United States

Ab124744 is a primary antibody that recognizes Histone H3 (phospho S10). It is a rabbit polyclonal antibody that can be used in various applications such as Western Blotting and Immunohistochemistry.

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Lab products found in correlation

6 protocols using ab124744

Comprehensive Protein Extraction and Analysis

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The cultured cells or tissues were harvested and used to extract total proteins by RIPA lysis (KCD-M1013, Cronda). The concentration of the total protein from each treatment was detected using Bicinchoninic acid assay (BL521A, Biosharp). Then equal protein was used to load on 12% Sodium dodecyl sulfate polyacrylamide gel electropheresis. The proteins were transferred to Polyvinylidene fluoride membranes after separation. Subsequently, the membranes were sealed by 5% non-fat milk for 2 hours and incubated with primary antibodies anti-IRF2 (1 : 1000, abcam, ab124744), anti-CD6 (1 : 2000, abcam, ab109217), anti-CD9 (1 : 5000, abcam, ab236630), anti-TSG101 (1 : 5000, abcam, ab125011), anti-caspase 3 (1 : 2000, abcam, ab32351), and anti-actin (1 : 2000, Serivicebio, GB12001) for one night at 4°C. The membrane was cultured by secondary antibody (1 : 4,000, BL003A, biosharp) for 1 hour at 37°C. Finally, the protein band was imaged by enhanced chemiluminescence (ECL) in a darkroom. Image J was used to detect the relative grey density.

Yuan Y., Tan S., Wang H., Zhu J., Li J., Zhang P., Wang M, & Zhang F. (2023). Mesenchymal Stem Cell-Derived Exosomal miRNA-222-3p Increases Th1/Th2 Ratio and Promotes Apoptosis of Acute Myeloid Leukemia Cells. Analytical Cellular Pathology (Amsterdam), 2023, 4024887.

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Protein Expression Analysis in Cell Lines

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HK-2 cells and animal tissues were lysed using RIPA lysis buffer (Thermo Fisher Scientific, MA, USA). Each protein sample was electrophoresed by 12% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Sigma-Aldrich). Next, the membranes were blocked with 5% bovine serum albumin (BSA) at room temperature for 2 h and incubated with the following primary antibodies: anti-IRF2 (ab124744, 1:1000, Abcam); anti-Bax (ab32503, 1:1000, Abcam); anti-Bcl-2 (ab196495, 1:1000, Abcam); anti-caspase-1 (ab62698, ab138483, 1:1000, Abcam); anti-caspase-4 (4450, 1:1000, Cell Signaling Technology, MA, USA), anti-caspase-11 (ab246496, 1:1000, Abcam); anti- apoptosis-associated speck-like protein (ASC; 13833, 67824, 1:1000, Cell Signaling Technology); anti-TNF-α (3707, 1:500, Cell Signaling Technology); anti-IL-1β (12242, 1:500, Cell Signaling Technology); anti-IL-18 (54943, 57058, 1:500, Cell Signaling Technology); anti-IL-6 (ab259341, 1:500, Abcam); anti-GSDMD (39754, 1:1000, Cell Signaling Technology); anti-NLRP3 (13158, 15101, 1:1000, Cell Signaling Technology); and GAPDH (ab8245, 1:3000, Abcam) overnight at 4 °C. Each membrane was incubated with corresponding secondary antibodies (7233, 7076, 1:5000, Cell Signaling Technology) for 1 h at room temperature. Protein expression was detected using an enhanced chemiluminescence detection kit (Thermo Fisher Scientific).

Zhang Y., Zhang Y., Yang A, & Xia F. (2021). Downregulation of IRF2 Alleviates Sepsis-Related Acute Kidney Injury in vitro and in vivo. Drug Design, Development and Therapy, 15, 5123-5132.

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Western Blot Analysis of Osteogenic Markers

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The experiment was performed as previously described.¹⁴ First, RIPA buffer was prepared, and cells were lysed for 30 min on ice. Then, the cell lysates were collected and centrifuged at 14,000 rpm for 30 min at 4°C. After that, the protein concentrations were measured, and equal amounts of proteins were mixed with loading buffer. The proteins were separated via electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, IPVH0010). Then the membranes were blocked with 5% non‐fat milk solution for 1 h. Thereafter, the membranes were incubated with primary antibodies followed by secondary antibodies. Finally, protein levels were detected using Chemiluminescent HRP Substrate (Millipore, WBKLS0500) and analyzed with ImageJ.
The following antibodies were used for western blotting: anti‐Osterix (Abcam, ab209484, 1:800); anti‐OCN (Abcam, ab93876, 1:800); anti‐AGO2 (Abcam, ab186733, 1:1500); anti‐RUNX2 (Cell Signaling Technology, 12556S, 1:1000); anti‐Collagen I (ColI; Abcam, ab260043, 1:1500); anti‐IRF2 (Abcam, ab124744, 1:2000); anti‐YY1 (Abcam, ab109228, 1:2000); anti‐GAPDH (CWBIO, CW0100M, 1:3000); and HRP‐conjugated secondary antibodies (Boster, 1:5000).

Ye G., Wang P., Xie Z., Li J., Zheng G., Liu W., Cao Q., Li M., Cen S., Li Z., Yu W., Wu Y, & Shen H. (2021). IRF2‐mediated upregulation of lncRNA HHAS1 facilitates the osteogenic differentiation of bone marrow‐derived mesenchymal stem cells by acting as a competing endogenous RNA. Clinical and Translational Medicine, 11(6), e429.

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Immunoblotting of NF-κB and Related Proteins

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The cell lysates were fractioned by 10% SDS-PAGE and then blotted onto nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). The membranes were incubated overnight with primary antibodies at a dilution of 1:1000, with the exception of anti-β-tubulin (1:2000). The antibodies used for immunoblotting were: rabbit anti-NF-κB p50 (sc-7178, Santa Cruz), rabbit NF-κB p65 (sc-7151, Santa Cruz), rabbit anti-Bcl-3 (sc-185, Santa Cruz), rabbit anti-STAT3 (sc-482, Santa Cruz), rabbit anti-IRF2 (ab124744, Abcam), rabbit anti-IκBα (9242S, Cell Signaling Technology, Danvers, MA, USA), mouse anti-phospho-IκBα (5A5, Cell Signaling), mouse anti-β-tubulin (T8328, Sigma-Aldrich, St. Louis, MO, USA), and mouse anti-lamin A+C (ab8984, Abcam). The membranes were then incubated with IRDye700-labeled donkey anti-mouse or anti-rabbit, or IRDye800-labeled donkey anti-mouse (LI-COR Biosciences) at a 1:5000 dilution. The blots were detected using an Odyssey^® Infrared Imaging System (LI-COR Biosciences).

Verhoeven R.J., Tong S., Zong J., Chen Y., Tsao S.W., Pan J, & Chen H. (2018). NF-κB Signaling Regulates Epstein–Barr Virus BamHI-Q-Driven EBNA1 Expression. Cancers, 10(4), 119.

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Western Blot Analysis of IRF1/2

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Whole cell lysates were prepared in RIPA buffer with protease inhibitor (Pierce), protein concentrations were determined by BCA assay (Pierce), and 10μg of denatured samples were run on 10% reducing gels (Genscript). After transfer, PVDF membranes (Millipore) were blocked with TBS-Tween 1x + 5% milk and then blotted with rabbit anti-IRF2 (Abcam ab124744) or rabbit anti-IRF1 (Abcam ab186384) in TBS-Tween 1x + 2% milk overnight at 4°C. The following day, membranes were washed 3x with TBS-Tween 1x, goat-anti-rabbit HRP (Millipore) was added for 1hr at RT, membranes were washed 3x, and HRP substrate (Millipore) was added. Following exposure, membranes were stripped (Millipore), blocked, and re-blotted with mouse anti-β-actin (Santa Cruz sc-47778) in TBS-Tween 1x + 2% milk overnight at 4°C. The following day, membranes were prepared as above except anti-mouse HRP (Pierce) was used instead.

Kriegsman B.A., Vangala P., Chen B.J., Meraner P., Brass A.L., Garber M, & Rock K.L. (2019). Frequent loss of IRF2 in cancers leads to immune evasion through decreased MHC-I antigen presentation and increased PD-L1 expression. Journal of immunology (Baltimore, Md. : 1950), 203(7), 1999-2010.

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Western Blot Analysis of IRF2 in H1299 and A549 Cells

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H1299 and A549 cells were lysed with RIPA buffer (Biosharp, Hefei, China) containing protease inhibitor PMSF (Biosharp, Hefei, China) to extract the total protein. Total protein concentration was determined by the BCA kit (Beyotime, Shanghai, China). Proteins were separated after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA). The PVDF membranes were then blocked with 5 % skimmed milk, and incubated with primary antibody (anti-IRF2, ab124744, Abcam, Shanghai, China, 1:1000) overnight at 4 °C, and secondary antibodiy (ab150077, Abcam, Shanghai, China, 1:2000) for 1 h at room temperature, respectively. At last, Enhanced Chemiluminescence Western Blotting Substrate (Biozym, Hessisch Oldendorf, Germany) was utilized to develop the protein bands.

Ma J., Miao H., Zhang H., Ren J., Qu S., Da J., Xu F, & Zhao H. (2021). LncRNA GAS5 modulates the progression of non-small cell lung cancer through repressing miR-221-3p and up-regulating IRF2. Diagnostic Pathology, 16, 46.

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