Lysates from the cells and microdissected renal tubules from each experimental group were separated in parallel on two 10% denaturing sodium dodecyl sulfate-polyacrylamide gels, transferred onto nitrocellulose membranes, blocked with 5% nonfat milk in 0.1% tris buffered saline with Tween-20 (TBST), and probed using antibodies at 4°C overnight. Primary antibodies against PPARγ (1:100, ab19481), TGF-β1 (1:100, ab27969), total Smad3 (1:100, ab40854), Smad3 (phospho S213) (1:100, ab63403), CTGF (1:100, ab6992), Fibronectin (1:200, ab2413), Collagen I (1:200, ab6308) and beta Actin (1:200, ab6276) were purchased from Abcam (Cambridge, USA). After extensive washing in TBST buffer, the secondary antibody (horseradish peroxidase-labeled IgG anti-rabbit/mouse antibody, Invitrogen, Cambridge, MA) was used at 1:3000 dilution for 1 hour at room temperature. The supersignal-enhanced chemoluminescent substrate (Pierce Biotechnology, Inc., Rockford, IL) was applied to the probed membrane and exposed for 10 minutes before the protein bands were visualized on radiograph films (Super Rx, Fuji Photo Film, Tokyo). Quantification was performed by measurement of the intensity of the bands using ImageJ analysis software (National Institutes of Health, Bethesda, MD).
Ab63403
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab63403 is a primary antibody product from Abcam. It is a rabbit monoclonal antibody that specifically recognizes the target protein. The antibody is suitable for use in various immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab53100, by Abcam (4 mentions)
Ab40854, by Abcam (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Ab92486, by Abcam (2 mentions)
Ab205718, by Abcam (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ab19481, by Abcam (1 mentions)
Ab27969, by Abcam (1 mentions)
Ab6992, by Abcam (1 mentions)
Horseradish peroxidase labeled igg anti rabbit mouse antibody, by Thermo Fisher Scientific (1 mentions)
Ab2413, by Abcam (1 mentions)
Ab6308, by Abcam (1 mentions)
Super rx, by Fujifilm (1 mentions)
Supersignal enhanced chemoluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Ab6276, by Abcam (1 mentions)
Ab39184, by Abcam (1 mentions)
Ab53154, by Abcam (1 mentions)
Ecl kit, by Merck Group (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
8 protocols using ab63403
1
Western Blot Analysis of Renal Fibrosis Markers
2
Western Blot Analysis of Protein Markers
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Total protein was isolated from treated HepG2 and HCCLM3 cells using RIPA lysis buffer containing protease inhibitors (Roche, Indianapolis, IN, USA). The protein concentrations across samples were measured using BCA protein assay kit (Beyotime, Shanghai, China). Equal amounts (20 μg) of protein were separated by 10% SDS-PAGE and then transferred onto PVDF membranes (EMD Millipore). After blocking with 5% skim milk, the membranes were incubated at 4°C overnight with primary antibodies against TIMP3 (ab39184; 1 : 1,000), TGF-β1 (ab9785; 1 : 200), p-smad3 (ab63403; 1 : 500), cleaved caspase-3 (ab2302), Bax (ab53154), Bcl-2 (ab196495), and β-actin (ab8227; 1 : 2,000) all from Abcam (Cambridge, MA, UK), followed by HRP-conjugated goat anti-rabbit IgG (ab7090; 1 : 10,000) at room temperature for 1 h. Signals were detected using an ECL kit (Millipore). β-actin served as an endogenous reference.
3
Western Blot Analysis of Inflammatory and TGF-β Signaling
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The expression of NLRP3, ASC, caspase-1, IL-1β, Smad2, Smad3, p-Smad2, p-Smad3 and TGF-β in tissues and cells was detected by Western blotting. Total protein was extracted from BRL-3A cells and the left liver lobe tissues and quantified by the BCA protein analysis kit. The proteins were separated by SDS-PAGE and transferred to the PVDF membrane. The GAPDH gene acts as a reference gene. Subsequently, the membrane was sealed with 5% skim milk powder and incubated overnight with primary antibodies NLRP3 (ab214185, 1: 500, Abcam, UK), ASC (ab168811, 1: 2000, Abcam, UK), caspase-1 (ab62698, 1: 500, Abcam, UK), IL-1β (ab2105, 1: 1000, Abcam, UK), Smad2 (ab40855, 1: 2000, Abcam, UK), Smad3 (ab40854, 1: 1000, Abcam, UK), p-Smad2 (ab53100, 1: 500, Abcam, UK), p-Smad3 (ab63403, 1: 500, Abcam, UK) and TGF-β (ab92486, 0.5–4 µg/mL, Abcam, UK) at 4°C.The next day, after washing with TBST for 3 times, the membrane was incubated with horseradish peroxidase-labeled secondary antibody at room temperature for 1 h. Proteins were visualized using enhanced chemiluminescence kits and gel imaging systems. The results are analyzed by Image Tools.
4
Protein Isolation and Western Blot Analysis
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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).
5
Western Blot Analysis of TGF-β Pathway Proteins
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Samples were lysed in RIPA buffer containing PMSF. The protein quantification was determined by BCA protein assay (Cat No. P0068, Beyotime, China), and equal amounts of proteins (40 μg) were subjected to SDS/PAGE (12% gels). After electrophoresis, proteins were transferred onto PVDF membranes (0.2 mm) in running buffer with 20% methanol. Non-specific sites were blocked with 5% (w/v) non-fat dried skimmed milk powder in TBST (2M Tris-HCl buffer, pH 7.6; 0.05 M NaCl; and 0.05% Tween-20) for 60 min at 37°C. The membranes were then incubated overnight at 4°C with the following antibodies, which were diluted in TBST: anti-TGF-β (product code: ab64715, Abcam, USA), anti-Smad2/3 (product code: ab236030, Abcam, USA), phosphorylated Smad2 (product code: ab53100, Abcam, USA), and phosphorylated Smad3 (product code: ab63403, Abcam, USA). After 4 washes in TBST, the membranes were incubated with horseradish-peroxidase-conjugated secondary antibodies (Cat No. A0562, Beyotime) for 1 h in TBST (dilution of 1: 5000). Protein bands were visualized by using an Enhanced Chemiluminescence (ECL) Plus Western blotting detection kit (Cat. No. P0018-2, Beyotime).
6
Protein Expression Analysis in Synovial Tissues
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Protein was extracted from the synovial tissues and MH7A cells using a protein extraction kit (Boster, Wuhan, China) and quantified with the BCA kit (Thermo Fisher Scientific, USA). Equal amounts of protein were mixed with the loading buffer, boiled for 15 min at 95°C, and loaded into a 10% SDS-PAGE gel. After resolving the proteins by SDS-PAGE, the bands were transferred to PVDF membranes. Then, they were blocked for 1 h with 5% skimmed milk powder at room temperature and incubated overnight with primary antibodies against p-Smad2 (ab53100, Abcam, UK), p-Smad3 (ab63403, Abcam, UK), Smad7(ab216428, Abcam, UK), TGF-β1 (ab92486, Abcam, UK), and β-actin (ab8226, Abcam, UK). The blots were washed and incubated with goat anti-rabbit IgG H&L (ab205718, Abcam, UK) secondary antibody. The protein bands were visualized using an ECL chemiluminescence kit (EMD Millipore, Germany) and quantified using Image-ProPlus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA).
7
Western Blot Analysis of Protein Expression
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The total protein was isolated from cells or tissues using RIPA buffer (Abcam) supplemented with protease and phosphatase inhibitor. The total protein was measured using the BCA protein assay Kit (Thermo Fisher Scientific). 20 µg of protein was separated with SDS-PAGE and transferred onto nitrocellulose membranes (Millipore). Subsequently, the membranes were blocked with 5% skim milk and incubated with primary antibodies overnight at 4 °C. Afterward, the membranes were incubated with the corresponding anti-mouse or anti-rabbit IgG secondary antibodies for 2 h at 37 °C, and then the protein bands were visualized by ECL reagent (Sigma-Aldrich). The gray value was determined using Image J analysis software. All the antibodies used in this study were purchased from Abcam (CD63, ab216130; TSG101, ab125011; CD9, ab223052; collagen I, ab260043; α-SMA, ab5694; fibronection, ab2413; METTL3, ab240595; Sp1, ab227383; TGF-β1, ab215715; p-Smad2, ab280888; p-Smad3, ab63403; Smad2, ab33875; Smad3, ab40854; GAPDH, ab8245).
8
Kidney Protein Expression Analysis
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First, the kidney tissues were homogenized. Then, proteins were extracted by using RIPA lysis buffer (Abcam, Ab156034, UK). The protein concentration was determined by a Pierce™ BCA Protein Assay Kit (Thermo Scientific). The protein extracts were separated by SDS–PAGE electrophoresis and then transferred onto PVDF (polyvinylidene fluoride) membranes. The membrane was blocked with 5% skim milk powder (diluted with Tris-buffer) for 2 h and then incubated overnight at 4 °C with primary antibodies against α-SMA (ab7817, 1:1000, Abcam, USA), ED-1 (qy-1088R, 1:1000, Santa Cruz, USA), TGF-β1 (ab215715, 1:500, Abcam, USA), p-Smad2 (ab280888, 1:1000, Abcam, USA), and p-Smad3 (ab63403, 1:1000, Abcam, USA). Subsequently, a secondary goat anti-rabbit IgG (HRP) antibody (ab205718, 1:2000, Abcam, USA) was applied and incubated with the membranes at room temperature for 1 h. The blots were assayed by using the Novex™ ECL Chemiluminescent Substrate Reagent Kit (Thermo Fisher Scientific™, WP20005, USA). β-actin was used as an internal reference protein. Finally, ImageJ software was used to analyze the results.
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