Ab10444

Manufactured by Abcam

Sourced in United Kingdom

Ab10444 is a recombinant monoclonal antibody produced in mouse. It is directed against the human CD4 antigen. The antibody is purified and conjugated with Alexa Fluor® 647.

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Lab products found in correlation

5 protocols using ab10444

Western Blot Analysis of ER Stress Markers

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Following DEV-CSC infection DEF cells were treated with radioimmunoprecipitation assay buffer (RIPA, Beyotime, China) containing phenylmethylsulfonyl fluoride (PMSF) at a ratio of 100 to 1 on ice. After 20 min of centrifugation, the total proteins were harvested and quantitatively analyzed using bicinchoninic acid (Beyotime Institute of Biotechnology, Nanjing, China). Each well of a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel were loaded with 50 µg of total protein, electrophoresed and then transferred to polyvinylidene difluoride membranes (PVDF; Millipore, Billerica, MA, USA). After blocking with 5% (w/v) nonfat milk, the proteins were incubated with primary antibodies at 4°C overnight. The next day, the proteins were labeled with a secondary antibody for 1 hr and the chemical signals were evaluated using the enhanced chemiluminescence detection kit (Beyotime) on the Tanon-5500 imaging system. The quantitative data were analyzed using Image J software. The primary antibodies used were as follows: CHOP (1:250; Abcam, Cambridge, UK, ab10444), GRP78 (1 µg/ml; Abcam, ab21685), ATF6 (1:300; Abcam, ab135707), and GAPDH (0.5 µg/ml; Abcam, ab37168). The secondary antibody was goat anti-mouse IgG H&L (1:1,000; Abcam, ab97040).

ZHANG Y., WANG X., HU A., WU Y., ZHANG P., YANG X., WEN Z, & WEN M. (2020). Duck enteritis virus infection suppresses viability and induces apoptosis and endoplasmic reticulum stress in duck embryo fibroblast cells via the regulation of Ca2+. The Journal of Veterinary Medical Science, 83(3), 549-557.

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Investigating ER Stress-Induced Apoptosis Signaling

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Cells were lysed by RIPA lysis (P0013B, Beyotime, China) supplied with PMSF and the lysates were quantitated by Bio-Rad DC Protein Assay kit (Ewell, China). The protein sample was separated using freshly-prepared SDS-PAGE, electrotransferred onto PVDF membranes, and probed with primary antibodies. After that, the membranes were re-probed with goat anti-rabbit IgG (1:10,000, ab6721, Abcam). Immunoblots were visualized with enhanced chemiluminescence detection reagents and captured under the SmartView Pro 2000 (UVCI-2100, Major Science, USA) microscope. Gray value of target protein bands was quantified using Image J software, with GAPDH used for normalization. Primary antibodies used: GRP78 (Abcam, ab21685, rabbit, 1:2000), IRE1 (Abcam, ab37073, rabbit, 1:1000), CHOP (Abcam, ab10444, rabbit, 1:1000), eIF2α (Abcam, ab169528, rabbit, 1:1000), cleaved-caspase3 (Abcam, ab32042, rabbit, 1:1000), caspase3 (Abcam, ab13847, rabbit, 1:500) and caspase 12 (Abcam, ab13847, 1:500).

Zhou N., Qiao H., Zeng M., Yang L., Zhou Y, & Guan Q. (2020). Circ_002117 binds to microRNA-370 and promotes endoplasmic reticulum stress-induced apoptosis in gastric cancer. Cancer Cell International, 20, 465.

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Investigating Inflammatory Mechanisms in Atherosclerosis

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Synthetic human IMD_1-53 was from Phoenix Pharmaceuticals (Burlingame, CA, USA). Alzet Mini-osmotic Pumps (model 2006) were from DURECT Corp (Cupertino, CA, USA). Primary antibodies for IMD (sc-86272), β-actin (sc-47778), IL-1β (sc-7884), and all horseradish peroxidase (HRP)-conjugated secondary antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Primary antibodies for CD68 (ab125212), α-actin (ab5694), glucose-regulated protein78 (GRP78, ab21865), activating transcription factor (ATF4, ab216839), cleaved ATF6 (ab203119), p-inositol-requiring kinase 1 alpha (p-IRE1α, ab48187), CHOP (ab10444), cleaved-caspase-3 (ab13847) were from Abcam PLC (Cambridge, UK). Apoptosis-associated speck-like protein containing CARD (ASC, SAB4501315) were from Sigma-Aldrich (St. Louis, MO, USA). Dylight-labeled secondary antibodies were from EarthOx Life Sciences (Millbrae, CA, USA). The kit for reverse transcription of RNA and SuperReal PreMix Plus for real-time PCR (TIANGEN Biotech, Beijing, China). The oil red O and taurine (Tau) were from Sigma-Aldrich (St. Louis, MO, USA). Oxidized human LDL (ox-LDL, YB-002) was from Yiyuan Biotechnology (Guangzhou, China). Other chemicals and reagents were of analytical grade.

Ren J.L., Chen Y., Zhang L.S., Zhang Y.R., Liu S.M., Yu Y.R., Jia M.Z., Tang C.S., Qi Y.F, & Lu W.W. (2021). Intermedin1-53 attenuates atherosclerotic plaque vulnerability by inhibiting CHOP-mediated apoptosis and inflammasome in macrophages. Cell Death & Disease, 12(5), 436.

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Immunoprecipitation and Western Blot Analysis of Protein Interactions

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Immunoprecipitation was performed on protein lysates from near-confluent cells. The cells were washed twice with ice-cold phosphate-buffered saline (PBS) and harvested by scraping in ice-cold lysis buffer containing 15 mM Tris (pH 8.0), 100 mM NaCl, 1% Triton X-100, supplemented with protease inhibitor cocktail (P2714, Sigma-Aldrich, St. Louis, MO). Lysates were incubated on ice for 30 min, clarified by centrifugation at 4°C, followed by protein estimation using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Overnight immunoprecipitation with anti-DDIT3 (sc-793, Santa Cruz Biotechnology, Dallas, TX) was performed using Dynabeads® Protein G (Life Technologies, Carlsbad, CA) as per manufacturer instructions. Next, SDS-PAGE separation and western blot were performed using anti-FUS (sc-373698, Santa Cruz) and anti-DDIT3 (ab10444, Abcam, Cambridge, UK) for detection. The following Cell Signaling Technology (Danvers, MA) antibodies were also used for western blot: anti-AKT (#2920), anti-phospho-AKT (Ser473) (#9271), anti-AXL (#4566) and PTEN (#9559). Antibodies of Abcam were used for western blot analysis of HDAC1 (ab19845), HDAC2 (ab32117) and HDAC3 (ab32369).^{38 (link)}

de Graaff M.A., Yu J.S., Cheung H.C., Ingram D.R., Nguyen T., Liu J.J., Bolshakov S., Szuhai K., Åman P., Torres K.E., Lev D., Nielsen T.O., Bovée J.V., Lazar A.J, & Somaiah N. (2016). Establishment and characterization of a new human myxoid liposarcoma cell line (DL-221) with the FUS-DDIT3 translocation. Laboratory investigation; a journal of technical methods and pathology, 96(8), 885-894.

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Rat Heart Protein Expression Analysis

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Proteins were extracted from the rat heart tissues using RIPA lysis buffer (Beyotime, Haimen, China). Protein concentrations were measured using a BCA protein assay kit (23227, Abcam). Post protein separation on 12% sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE), the separated protein was transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were then blocked with 5% non-fat milk at 4 °C for 1 hour, and incubated with the following primary antibodies: DDIT3 antibody (ab10444, Abcam), cleaved caspase-12 antibody (#2202, CST, MA, USA), GADD34 antibody (ab131402, Abcam), BiP antibody (ab21685, Abcam), cleaved caspase-3 antibody (ab184784, Abcam), cleaved caspase-9 antibody (#9507, CST), IL-1β (205924, Abcam) p-ERK1/2 antibody (ab214362, Abcam), ERK1/2 antibody (ab17942, Abcam), P65 antibody (ab16502, Abcam), and p-P65 (phospho S536) antibody (ab86299, Abcam). After incubation overnight, the membranes were supplemented with the corresponding secondary antibodies. The bands were visualized with ECL reagent (Bio-Rad, CA, USA). All experiments were performed at least three times.

Chen H., Shi Z., Xing Y., Li X, & Fu F. (2020). Fangchinoline attenuates cardiac dysfunction in rats with endotoxemia via the inhibition of ERK1/2 and NF-κB p65 phosphorylation. Annals of Translational Medicine, 8(18), 1167.

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