Ab9482

Manufactured by Abcam

Sourced in United States, United Kingdom

Ab9482 is a laboratory product manufactured by Abcam. It is a tool for scientific research and experimentation. The core function of this product is to facilitate laboratory procedures, but a detailed description cannot be provided while maintaining an unbiased and factual approach.

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Lab products found in correlation

Ripa lysis buffer, by Beyotime (7 mentions) β actin, by Cell Signaling Technology (6 mentions) Ab92547, by Abcam (5 mentions) Ab40776, by Abcam (5 mentions) Ab182651, by Abcam (5 mentions) Ripa buffer, by Beyotime (4 mentions) Ab32503, by Abcam (4 mentions) Chemiluminescence chromogenic substrate, by Beyotime (4 mentions) Ab181602, by Abcam (4 mentions) Ab15148, by Abcam (4 mentions) Ab68418, by Abcam (3 mentions) Polyvinylidene difluoride membrane, by Merck Group (3 mentions) Ab134175, by Abcam (3 mentions) Ab135366, by Abcam (3 mentions) Ab104836, by Abcam (3 mentions) Ab32042, by Abcam (2 mentions) Ab182858, by Abcam (2 mentions) Ab692, by Abcam (2 mentions) Ab125011, by Abcam (2 mentions) Sds page gel, by Ipsen (2 mentions)

Close spelling variants of this product

Ab9482-200 (2 citations)

48 protocols using ab9482

Extraction and Characterization of MO-B from Ophiopogon japonicus

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Ophiopogon japonicus was obtained from farms in Cixi (Zhejiang, China). MO-B was extracted from Ophiopogon japonicus using high-speed counter-current chromatography (19 (link)) and the yield was ~0.2–0.4 mg/g in tuber roots of Ophiopogon japonicus. High-performance liquid chromatography (HPLC) was conducted to measure the purity of MO-B, which was >97% (Fig. S1).HPLC was performed using a Shimadzu C18 column (5 µm 250×4.6 mm). The volume ratio of mobile solvents A (water) and B (acetonitrile) was maintained at 35:65, and the temperature was set at 30°C. The flow rate of the mobile phase was 1 ml/min. The detection wavelength was 285 nm. MO-B was then dissolved to 10, 20, 40 and 50 µM in dimethyl sulfoxide for cell treatment.
Antibodies targeting Bax (ab182733), Bcl-2 (ab182858), cleaved caspase-3 (ab32042), neutrophil cytochrome b light chain (p22phox; ab80896) and GAPDH (ab9482), goat anti-mouse horseradish peroxidase IgG (ab6789) and goat anti-rabbit IgG horseradish peroxidase (ab6721) secondary antibodies, were purchased from Abcam. Cell Counting Kit-8 (CCK-8), ROS and malondialdehyde (MDA) detection kits, radioimmunoprecipitation assay (RIPA) lysis buffer, a BCA Protein Assay kit and superoxide dismutase (SOD) assay kit with WST-8 were purchased from Beyotime Institute of Biotechnology.

Wang L., Zhou Y., Qin Y., Wang Y., Liu B., Fang R, & Bai M. (2019). Methylophiopogonanone B of Radix Ophiopogonis protects cells from H2O2-induced apoptosis through the NADPH oxidase pathway in HUVECs. Molecular Medicine Reports, 20(4), 3691-3700.

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Protein Extraction and Western Blot Analysis

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Proteins were extracted using RIPA lysis buffer (Beyotime) and quantified by bicinchoninic acid (BCA) method following the standard protocol. An equal amount of extracts was treated with 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and shifted onto a nitrocellulose membrane (Millipore). Subsequently, the membranes were incubated with specific primary antibodies PAX6 (1:1000, ab5790, Abcam) and GADPH (1:1000, ab8245, Abcam), and followed by interaction with secondary HRP-conjugated antibody (1:1000, ab9482, Abcam). Finally, protein signals were examined using an ECL method.

Jiang Q., Xing W., Cheng J, & Yu Y. (2020). Knockdown of lncRNA XIST Suppresses Cell Tumorigenicity in Human Non-Small Cell Lung Cancer by Regulating miR-142-5p/PAX6 Axis. OncoTargets and therapy, 13, 4919-4929.

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Exosome and Cell Protein Analysis

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Both exosomes and cells were lysed using the radio-immunoprecipitation assay (RIPA) buffer (Beyotime, Shanghai, China). Cell lysates were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, shifted onto a polyvinylidene fluoride membrane, and blocked with 5% non-milk. Next, the membranes were incubated with primary antibodies against CD9 (1:5000, ab68418, Abcam, Cambridge, MA, USA), CD63 (1:2000, ab68418, Abcam), Matrix metallopeptidase 9 (MMP-9) (1:1000, ab38898, Abcam), Vimentin (1:5000, ab92547, Abcam), B-cell lymphoma-2 (Bcl-2) (1:1000, ab692, Abcam), BCL2-associated X protein (Bax) (1:1000, ab32503, Abcam), and glyceraldehyde 3-phosphate dehydrogenase (GADPH) (1:10,000, ab181602, Abcam), followed by interaction with secondary HRP-conjugated antibody (1:1000, ab9482, Abcam). Finally, signals were visualized with the chemiluminescence chromogenic substrate (Beyotime).

Rong F., Liu L., Zou C., Zeng J, & Xu Y. (2020). MALAT1 Promotes Cell Tumorigenicity Through Regulating miR-515-5p/EEF2 Axis in Non-Small Cell Lung Cancer. Cancer Management and Research, 12, 7691-7701.

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Western Blot Analysis of Protein Markers

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Total protein was extracted from the brain tissue from the rats in each study group using RIPA buffer (Santa Cruz Biotechnology, Inc.) containing a protease inhibitor (Sigma-Aldrich, St. Louis MO, USA). A bicinchoninic acid (BCA) assay kit (Beyotime, Shanghai, China) was utilized for conducting quantitative analysis of protein concentrations. About 40 μg protein sample was separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and electrophoresis and then transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA). After treating with 5% skimmed milk, primary and secondary antibodies were utilized to incubate the membranes for 1.5 h. Enhanced chemiluminescence (ECL) (Bio-Rad, Hercules, CA, USA) was used to visualize the blots. Primary antibodies used were to Notch1 (1: 1000) (ab65297; Abcam, Cambridge, MA, USA), PTEN (1: 1000) (ab31392; Abcam, Cambridge, MA, USA), Nestin (1: 1000) (ab22035; Abcam, Cambridge, MA, USA), and GAPDH (1: 2000) (ab9482; Abcam, Cambridge, MA, USA).

Sha R., Han X., Zheng C., Peng J., Wang L., Chen L, & Huang X. (2019). The Effects of Electroacupuncture in a Rat Model of Cerebral Ischemia-Reperfusion Injury Following Middle Cerebral Artery Occlusion Involves MicroRNA-223 and the PTEN Signaling Pathway. Medical Science Monitor : International Medical Journal of Experimental and Clinical Research, 25, 10077-10088.

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Exosome Protein Characterization Protocol

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Proteins were extracted from cells or exosomes using RIPA lysis buffer (Beyotime, Beijing, China) and quantified and determined using a bicinchoninic acid Protein Assay Kit (Beyotime). Extractive protein was loaded on sodium dodecyl sulfate polyacrylamide gel electrophoresis for separation and then shifted onto polyvinylidene fluoride membranes. Later, membranes were interacted with CD81 (ab79559, 1:1,000, Abcam, Cambridge, MA, USA), CD63 (1:2,000, ab68418, Abcam), TSG101 (ab125011, 1:5,000, Abcam), HMGB3 (1:1,000, #6893, Cell Signaling Technology, Beverly, MA, USA), and the secondary HRP-conjugated antibody (1:1,000, ab9482, Abcam). The β-actin (1:1,000, #4970, Cell Signaling Technology) was used as an internal reference. The protein bands were visualized using the Image J software.

Yu Q., Li Y., Peng S., Li J, & Qin X. (2021). Exosomal-mediated transfer of OIP5-AS1 enhanced cell chemoresistance to trastuzumab in breast cancer via up-regulating HMGB3 by sponging miR-381-3p. Open Medicine, 16(1), 512-525.

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Protein Extraction and Immunoblotting Analysis

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Proteins were extracted using RNA immunoprecipitation (RIP) assay lysis buffer (Beyotime, Beijing, China) and quantified by bicinchoninic acid (BAC) method, then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and shifted onto a polyvinylidene fluoride (PVDF) membrane. Immunoblotting used antibodies against CD9 (1: 5000, ab68418, Abcam, Cambridge, MA, USA), CD63 (1: 2000, ab68418, Abcam), E-cadherin (E-cad; 1: 1000, ab15148, Abcam), vimentin (1: 5000, ab92547, Abcam), ERα (1: 3000, ab13504, Abcam), GAPDH (1: 10 000, ab181602, Abcam) and the secondary horseradish peroxidase (HRP)-conjugated antibody (1: 1000, ab9482, Abcam). The protein bands were visualized using electrochemiluminescence (ECL).

Hu K., Liu X., Li Y., Li Q., Xu Y., Zeng W., Zhong G, & Yu C. (2020). Exosomes Mediated Transfer of Circ_UBE2D2 Enhances the Resistance of Breast Cancer to Tamoxifen by Binding to MiR-200a-3p. Medical Science Monitor : International Medical Journal of Experimental and Clinical Research, 26, e922253-1-e922253-12.

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Western Blot Analysis of Protein Signaling

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Total protein lysates were prepared as previously described.3, 29, 30 Proteins were separated by SDS‐PAGE and transferred to the nitrocellulose membranes. Then the membranes were incubated with antibodies against CYP2E1 (Abcam; ab28146, 1:5000), phosphor‐p44/42 MAPK (Erk1/2) (Thr201/Tyr204) (Cell Signaling; 4370, 1:2000), p44/42 MAPK (Erk1/2) (Cell Signaling; 9102, 1:1000), phosphor‐PI3 Kinase p85 (Tyr458)/p55 (Tyr199) (Cell Signaling; 4228, 1:1000), PI3 Kinase p85 (Cell Signaling; 11889, 1:1000), phosphor‐Akt (Ser473) (Cell Signaling; 4060, 1:2000), Akt (Cell Signaling; 8596, 1:1000), Myc (Abcam; ab17355, 1:500), CEBP Alpha (Abcam, ab40764), CEBP Beta (Abcam, ab32358), or C/EBP zdta (ThermoFisher, PA5‐37925). Antibody binding was detected with horseradish peroxidase–conjugated immunoglobulin G using a chemiluminescence detection system after incubation with the appropriate secondary antibodies. GAPDH was used as normalization (Abcam; ab9482, 1:5000). Bands were quantitatively analyzed with ImageJ software.

Guan F., Yang X., Li J., Dong W., Zhang X., Liu N., Gao S., Wang J., Zhang L, & Lu D. (2018). New Molecular Mechanism Underlying Myc‐Mediated Cytochrome P450 2E1 Upregulation in Apoptosis and Energy Metabolism in the Myocardium. Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease, 8(1), e009871.

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Western Blot Analysis of BMPR1B and GAPDH

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Antibodies for BMPR1B (ab155058) and GAPDH (ab9482) were obtained from Abcam (UK). Western blotting was performed according to a previously reported method (Liu et al., 2016 (link)).

Abdurahman A., Aierken W., Zhang F., Obulkasim R., Aniwashi J, & Sulayman A. (2022). miR-1306 induces cell apoptosis by targeting BMPR1B gene in the ovine granulosa cells. Frontiers in Genetics, 13, 989912.

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Western Blot Protocol for Protein Analysis

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Protein lysate was used to lysate cells or tissues. The lysate consisted of RIPA buffer (Thermo Scientific, MA: Massachusetts, USA), phosphorylase inhibitor (Roche 5892791001, Basel, Switzerland), protease inhibitor (Roche 04693132001, Basel, Switzerland), etc. The concentration of the obtained protein was measured by the BCA kit of Biyuntian. Sodium dodecyl sulfate (SDS, CWBIO, Beijing, China) was added and denaturated at 100 °C for 20 min. The 8% and 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels (EpiZyme, Shanghai, China) were selected, and the sample size was 10 µL or 20 µL. After electrophoresis, the prefabricated adhesives were transferred to 0.45 μm Hybridization Nitrocellulose Filter (NC) membrane (Merck, New Jersey, USA), which was sealed with 5% skim milk powder, and then the primary and secondary antibodies were incubated. Primary antibodies Adamts5 (1:1000, Abcam ab41037, Cambridge, UK), KI67 (1:1000, Bioss bs-23102R, Beijing, China), PCNA (1:1000, Abcam ab18197, Cambridge, UK), MyHC (developmental myosin 1:1000, DSHB MF20, Iowa, USA) and Gapdh (1:1000, Abcam ab9482, Cambridge, UK) were diluted by 1× Tween (TBST) buffer (EpiZyme, Shanghai, China). Secondary antibodies were derived from rabbits and mice. ImageJ software (NIH, Bethesda, Maryland, USA) was used to analyze the gray value of protein bands.

Liu Y., Yao Y., Zhang Y., Yan C., Yang M., Wang Z., Li W., Li F., Wang W., Yang Y., Li X, & Tang Z. (2023). MicroRNA-200c-5p Regulates Migration and Differentiation of Myoblasts via Targeting Adamts5 in Skeletal Muscle Regeneration and Myogenesis. International Journal of Molecular Sciences, 24(5), 4995.

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Western Blot Analysis of Bcl-2 Pathway

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Transfected cells were lysed by RIPA lysis buffer (Beyotime). Then the lysate was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and transferred to a polyvinylidene fluoride membranes. Subsequently, the membranes were incubated with primary antibody against B-cell lymphoma-2 (Bcl-2) (1:1000, ab692, Abcam, Cambridge, MA, USA), BCL2-associated X protein (Bax) (1:1000, ab32503, Abcam), phosphorylated (p)-phosphoinositide 3-kinase (p-PI3K) (1:1000, ab182651, Abcam), PI3K (1:1000, ab40776, Abcam), p-protein kinase B (p-AKT) (1:1,000, 9271, Cell Signaling Technology, Boston, MA, USA), AKT (1:1,000, 9272, Cell Signaling Technology), as well as β-Actin (1:1,000, 4967, Cell Signaling Technology). After interaction with secondary HRP-conjugated antibody (1:1000, ab9482, Abcam). Experiments were performed three times, and immunoreactive bands were visualized using electrochemiluminescence.

Shen Q., Jiang S., Wu M., Zhang L., Su X, & Zhao D. (2020). LncRNA HEIH Confers Cell Sorafenib Resistance in Hepatocellular Carcinoma by Regulating miR-98-5p/PI3K/AKT Pathway. Cancer Management and Research, 12, 6585-6595.

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