Ab8425

Manufactured by Abcam

Sourced in United Kingdom

Ab8425 is a mouse monoclonal antibody that recognizes the protein CD4. It is intended for use in flow cytometry, immunohistochemistry, and other immunoassay applications.

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Lab products found in correlation

5 protocols using ab8425

Western Blot Analysis of Liver Proteins

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Livers were homogenized in RIPA buffer using a FastPrep-24 homogenizer and incubated on ice for 30 min. Samples were centrifuged at 17,000 x g for 15 min at 4 ^oC. Supernatants were resuspended in 2X SDS loading buffer and 25 or 100 µg of total protein was run on a 4-15% SDS-PAGE gel before being transferred to nitrocellulose membrane. Membranes were blocked for 1 hr in 10% milk TBS-T solution at room temperature before overnight incubation in anti-CA3 (Abcam, Cambridge, MA, catalog# ab181358, 1:500-1000) and anti-GAPDH (Abcam, catalog# ab8425, 1:5000) antibody in TBS-T at 4 ^oC. After washing, samples were incubated in goat anti-rabbit HRP secondary antibody (Thermo-Fisher, catalog # 656120, 1:2000) in 5% milk TBS-T solution for 3 hr before washing and visualization with ECL (Thermo-Fisher).

Bell-Temin H., Yousefzadeh M.J., Bondarenko A., Quarles E., Jones-Laughner J., Robbins P.D., Ladiges W., Niedernhofer L.J, & Yates N.A. (2019). Measuring biological age in mice using differential mass spectrometry. Aging (Albany NY), 11(3), 1045-1061.

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Western Blot Analysis of TGFBR1, CCNE2, and GAPDH

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For western blot analysis, RIPA buffer containing protease inhibitors and phosphatase inhibitors (Roche) was used to prepare whole-cell lysates. Equal amounts of proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes (Millipore). After blocking with 5% bovine serum albumin (BSA), the membranes were probed with anti-TGFBR1, CCNE2, and anti-GAPDH (ab31013, ab32103, and ab8425; Abcam, Cambridge, UK), followed by incubation with a horseradish peroxidase-conjugated secondary antibody goat-anti-mouse IgG (1:2000) and goat-anti-rabbit IgG (1:3000). Proteins were visualized using Image Reader LAS-4000 (Fujifilm) and analyzed using the Multi Gauge V3.2 software.

Gao P., Wang H., Yu J., Zhang J., Yang Z., Liu M., Niu Y., Wei X., Wang W., Li H., Wang Y, & Sun G. (2018). miR-3607-3p suppresses non-small cell lung cancer (NSCLC) by targeting TGFBR1 and CCNE2. PLoS Genetics, 14(12), e1007790.

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Protein Expression Analysis in Ischemic Mouse Brains

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Total protein of the cortex of the ischemia side in mice brains or cultured cells was extracted using a RIPA lysis buffer (Beyotime Institute of Biotechnology, China). Approximately equal amounts of protein were separated by 12% SDS-PAGE and transferred onto PVDF membranes. After blocking in 5% skim milk, the membranes were incubated with primary antibodies, including FGF19 (1:500, ab225942, Abcam), GSK-3β (1:500, ab93926, Abcam), phosphorylated-GSK-3β (1:500, ab75814, Abcam), Nrf2 (1:500, ab62352, Abcam), Lamin B2 (1:1000, ab8983, Abcam) and GAPDH (1:2000, ab8425, Abcam), overnight at 4 °C. Afterwards, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:3000, ab205718, Abcam) at room temperature for 1 h. The protein bands were visualized using an enhanced chemiluminescent (ECL) kit.

Zhou Y., Yang L., Bo C., Zhang X., Zhang J, & Li Y. (2021). MicroRNA-9-3p Aggravates Cerebral Ischemia/Reperfusion Injury by Targeting Fibroblast Growth Factor 19 (FGF19) to Inactivate GSK-3β/Nrf2/ARE Signaling. Neuropsychiatric Disease and Treatment, 17, 1989-2002.

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Western Blot Analysis of FOXO3 Protein

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For western blot analyses, RIPA buffer containing protease inhibitors and phosphatase inhibitors (Roche) was used to prepare whole-cell lysates. Briefly, equal amounts of lysate were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to PVDF membranes (Millipore). After blocking the membranes with 5% bovine serum albumin (BSA), they were probed with anti-FOXO3 and anti-GAPDH (ab12162, ab8425, Abcam, Cambridge, UK), followed by incubation with the horseradish peroxidase–conjugated secondary antibodies goat-anti-mouse IgG (1:2000) and goat-anti-rabbit IgG (1:3000). Proteins were visualized by Image Reader LAS-4000 (Fujifilm) and analyzed by Multi Gauge V3.2 software.

Lu Y.F., Yu J.R., Yang Z., Zhu G.X., Gao P., Wang H., Chen S.Y., Zhang J., Liu M.Y., Niu Y., Wei X.M., Wang W., Ye F.J., Zhang L.X., Zhao Y, & Sun G.G. (2018). Promoter hypomethylation mediated upregulation of MicroRNA-10b-3p targets FOXO3 to promote the progression of esophageal squamous cell carcinoma (ESCC). Journal of Experimental & Clinical Cancer Research : CR, 37, 301.

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Quantification of DNA Damage Response in Mouse Liver

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Snap-frozen livers from mice were incubated in RIPA buffer (Thermo-Fisher) on ice for 30 min after being homogenized with a FastPrep-24 homogenizer. Samples were centrifuged at 17,000g for 15 min at 4 °C. Supernatant was resuspended in 2× SDS loading buffer and 50 μg of total protein run on a 4–15% SDS–PAGE gel (Bio-Rad) before being transferred to nitrocellulose membrane. Membranes were blocked for 1 h in 10% milk TBS-T solution at room temperature before incubation in anti-ERCC1 (Santa Cruz Biotechnology, sc-17089), anti-γH2AX (Novus Biologicals, NB100-384, 1:2,000) and anti-GAPDH (Abcam, ab8425, 1:5,000) antibody at 4 °C overnight. After washing, samples were incubated in either horse anti-mouse HRP (Cell Signaling Technology, 7076S) or goat anti-rabbit HRP secondary antibody (Thermo-Fisher, 656120, 1:2,000) in 5% milk TBS-T solution for 3 h before washing and visualization with ECL (Thermo-Fisher).

Yousefzadeh M.J., Flores R.R., Zhu Y., Schmiechen Z.C., Brooks R.W., Trussoni C.E., Cui Y., Angelini L., Lee K.A., McGowan S.J., Burrack A.L., Wang D., Dong Q., Lu A., Sano T., O’Kelly R.D., McGuckian C.A., Kato J.I., Bank M.P., Wade E.A., Pillai S.P., Klug J., Ladiges W.C., Burd C.E., Lewis S.E., LaRusso N.F., Vo N.V., Wang Y., Kelley E.E., Huard J., Stromnes I.M., Robbins P.D, & Niedernhofer L.J. (2021). An aged immune system drives senescence and ageing of solid organs. Nature, 594(7861), 100-105.

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