Total protein was isolated from OS cells and xenograft tumor tissues by Radio Immunoprecipitation Assay lysis buffer. After separating by 10% sodium dodecyl sulfate polyacrylamide gel, proteins were then moved to polyvinylidene fluoride membranes (Millipore, United States). Then, the membranes were blocked with 5% nonfat milk, incubated with primary antibodies against GAPDH (ab181602, 1:10,000; Abcam, Shanghai, China), transforming growth factor-β1 (TGF-β1; ab179695, 1:1,000), SMAD2 (ab33875, 1:1,000), SMAD3 (ab40854, 1:1,000), Smad2 (phospho S255) (ab188334, 1:2,000), Smad2 (phospho S467) (ab53100, 1:500), Smad3 (phospho T179) (ab193297, 1:1,000), Smad3 (phospho S213) (ab63403, 1:1,000), Smad3 (phospho S423/S425) (ab52903, 1:2,000), and Ki67 (ab16667, 1:1,000) at 4°C overnight, followed by incubation with secondary antibody for 1 h at darkness. The relative levels of proteins were evaluated using an ECL chemiluminescent detection system (Thermo Fisher Scientific, Rochester. NY).
Ab193297
Manufactured by Abcam
Sourced in United Kingdom
Ab193297 is a lab equipment product. It serves as a core function in laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab40854, by Abcam (3 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Ab53100, by Abcam (1 mentions)
Ecl chemiluminescent detection system, by Thermo Fisher Scientific (1 mentions)
Ab52903, by Abcam (1 mentions)
Ab63403, by Abcam (1 mentions)
Ab33875, by Abcam (1 mentions)
Ab179695, by Abcam (1 mentions)
Ab188334, by Abcam (1 mentions)
Ab16667, by Abcam (1 mentions)
Ab181602, by Abcam (1 mentions)
Horseradish peroxidase linked anti mouse or anti rabbit igg, by Cell Signaling Technology (1 mentions)
Ab6308, by Abcam (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Ab6310, by Abcam (1 mentions)
Ab208182, by Abcam (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Boster Bio (1 mentions)
P mtor, by Cell Signaling Technology (1 mentions)
Bms630, by Thermo Fisher Scientific (1 mentions)
Bms622, by Thermo Fisher Scientific (1 mentions)
4 protocols using ab193297
1
Protein Isolation and Western Blot Analysis
2
Myocardial Protein Expression Analysis
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Fresh myocardiums were lysed in RIPA buffer containing 0.1% PMSF (BOSTER Biological Technology; Wuhan, China). Proteins were separated by SDS-PAGE using 8–10% gradient gel and transferred onto 0.45 μm PVDF membranes. The expressions of target protein were normalized to GAPDH protein. Antibodies against α-SMA (#19245), PTEN (#9188), p-Akt (#9271), Akt (#9272), p-mTOR (#5536), mTOR (#2972), GAPDH (#2118) and horseradish peroxidase-linked anti-mouse or anti-rabbit IgG (#7074) were purchased from Cell Signaling Technology (Boston, USA). Antibodies against collagen-I (ab6308), collagen-I (ab6310), p-Smad3 (ab193297) and Smad3 (ab208182) were purchased from Abcam (Cambridge, UK).
3
Moracin Modulates Inflammatory Response
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The drug, moracin (wkq-00871) was purchased from Sichuan Victory Biological Technology Co., Ltd (Chengdu, China). LPS (Escherichia coli O111:B4) was purchased from Sigma (St. Louis, MO, U.S.A.). Catalase (CAT, LE-06378), malondialdehyde (MDA, LE-07345) and superoxide dismutase (SOD, LE-07334) detection kits were bought from Lai Er Bio-Tech (Hefei, China). Enzyme-linked immunosorbent assay (ELISA) kits for IL-6 (CRC0063), TNF-α (BMS622) and IL-1β (BMS630) were obtained from eBioscience. CO., LTD. All the primary antibodies used in the present study, including antibodies for Nrf-2 (ab137550), HO-1 (ab13243), TGF-β (ab190503), p-smad-3 (ab193297), smad-3 (ab40854), p-IκBα (ab133462), IκBα (ab32518), p-NF-κBp65 (ab86299), NF-κBp65 (ab16502) were from Abcam (Cambridge, U.K.).
4
Protein Quantification and Western Blot Analysis
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Protein was extracted from tissues or cells by using RIPA lysate (Hat Biotechnology, China). After quantification with bicinchoninic acid, equal amounts of protein were loaded and separated by SDS-PAGE, then transferred electrophoretically to polyvinylidene difluoride membranes, which were incubated with primary antibodies overnight at 4, followed by a 1:5000 dilution of goat anti-rabbit IgG antibody (EK020, Zhuangzhi Biotech, China) at room temperature. Primary antibodies were for CTGF (1:1000, ab6992), Smad3 (1:1000, ab40854), Smad4 (1:1000, ab40759), p-Smad3 (1:500, ab193297), cyclin D2 (1:1000, ab230883), p21 (1:500, ab109199; all Abcam, England); EZH2 (1:500, 5246; CST, USA); Col I (1:500, WL0088), Col III (1:500, WL0318), β-actin (1:500, WL01372; all Wanleibio, China), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:1000, AF7021, Affinity Biosciences, USA). Protein bands were visualized by using a potent ECL kit (KF001, Affinity Biosciences, USA) and the Gene Company imaging system (China). GAPDH or β-actin was an internal reference.
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