An appropriate amount of RIPA lysate was used to lysate cells or tissues and extract proteins. Protein concentration was measured with BCA kit (Abcam, ab102536). Protein samples and protein markers were loaded in 10% separation gel and then transferred to 0.45-μm PVDF membranes (Millipore, 42029053) activated with methanol. Then membrane was blocked in 5% non-fat milk for 2 h and incubated with primary antibody at 4 °C overnight. Then placed into the corresponding secondary antibody (ab288151, ab6728, Abcam, USA,1:5000) box and incubated with a shaker at room temperature for 2 h. Finally, the ECL working solution (Thermo Scientific™, USA) was prepared according to 1:1, and the membrane was exposed in the chemiluminescence instrument, the results were analyzed by Image J. The involved primary antibodies were as follow and concentration was diluted according to product instructions: TPPP3 (NBP2-13469, NBP2-95209, Novus Biologicals), and N-cadherin (ab76011, Abcam), E-cadherin (ab40772, Abcam), Vimentin (ab8978, Abcam), Snail1 (13099-1-AP, Proteintech), Slug (ab302780, Abcam), Twist1 (ab175430, Abcam), ZEB1 (ab203829, Abcam), β-catenin(ab203829, Abcam).
Ab203829
Manufactured by Abcam
Sourced in United States, United Kingdom, China
Ab203829 is a primary antibody. It is a purified recombinant protein produced in E. coli.
Automatically generated - may contain errors
Lab products found in correlation
Ab40772, by Abcam (10 mentions)
Pvdf membrane, by Merck Group (8 mentions)
Ab8227, by Abcam (7 mentions)
Ab216347, by Abcam (7 mentions)
Ab92547, by Abcam (6 mentions)
Ab76011, by Abcam (5 mentions)
Polyvinylidene difluoride membrane, by Merck Group (5 mentions)
Ab181602, by Abcam (5 mentions)
Ab6721, by Abcam (5 mentions)
Ab8978, by Abcam (4 mentions)
Ab175430, by Abcam (4 mentions)
Ab180714, by Abcam (3 mentions)
Ab32042, by Abcam (3 mentions)
Ab1416, by Abcam (3 mentions)
Ab138222, by Abcam (3 mentions)
Ab179467, by Abcam (3 mentions)
Ab8805, by Abcam (3 mentions)
Ripa buffer, by Beyotime (3 mentions)
Ab8226, by Abcam (3 mentions)
Ripa lysis buffer, by Beyotime (3 mentions)
50 protocols using ab203829
1
Protein Expression Analysis Protocol
2
Quantitative Immunochemistry Staining Protocol
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Immunochemistry staining procedures were performed as described previously.20 (link),21 Briefly, expression levels of E-cadherin (1:500; ab40772, Abcam plc, USA), cytokeratin 19 (1:400), vimentin (1:100; AF7013, Affinity Biosciences, USA), ZEB1 (1:100; ab203829, Abcam plc, USA), and HSPB1 (1:100; ab203829, Abcam plc, USA) were determined using specific, commercially available antibodies. Control sections (incubated without the primary antibody) showed little to no immunostaining. Five digital images were collected from random areas of IHC slides using an optical microscope (BX51, Olympus, Japan) and ISCapture software (Tucsen, China). Immunostaining intensity was then quantified using Image Pro Plus v. 6.0 software (Media Cybernetics, USA).
3
Regulation of ZEB1 Ubiquitination by BRCC3 in Cancer Cells
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MDA-MB-231 cells were transfected with shRNA-BRCC3 or the negative control and were grouped into HA-ubiquitin (Ub), Flag-ZEB1+HA-Ub, Flag-ZEB1+shRNA+HA-Ub, or Flag-ZEB1+shRNA-BRCC3+HA-Ub groups. BT-549 cells were transfected with wild-type (WT) BRCC3 or mutant (Mut) BRCC3 and grouped into the following groups: Flag-ZEB1+HA-Ub, Flag-ZEB1+WT-BRCC3+HA-Ub, and Flag-ZEB1+Mut-BRCC3+HA-Ub. After MG132 (20 μM) was added, the cells were collected after 8 h and centrifuged at 4°C and 1600
g for 5 min. The supernatant was discarded, and the mixture was placed on ice. The cells were resuspended by adding 400 μL of HEPES buffer (Thermo Fisher, Waltham, USA), lysed by ultrasonication, and centrifuged at 4°C and 3000
g for 10 min. Then, 40 μL of supernatant was mixed with 2×loading buffer, and the expression of intracellular proteins was detected by western blot analysis using anti-ZEB1 antibody (ab203829, 1:500; Abcam). The remaining 360 μL of supernatant was added to 2 μL of anti-Flag antibody and incubated at 4°C for 4‒6 h. Then, 40–60 μL of protein A/G agarose was added and incubated at 4°C for 8–10 h. After centrifugation at 4°C and 1600
g for 3 min, supernatant was discarded, and the beads were mixed with 2× loading buffer and subject to western blot analysis using anti-ZEB1 antibody (ab203829, 1:500; Abcam).
g for 5 min. The supernatant was discarded, and the mixture was placed on ice. The cells were resuspended by adding 400 μL of HEPES buffer (Thermo Fisher, Waltham, USA), lysed by ultrasonication, and centrifuged at 4°C and 3000
g for 10 min. Then, 40 μL of supernatant was mixed with 2×loading buffer, and the expression of intracellular proteins was detected by western blot analysis using anti-ZEB1 antibody (ab203829, 1:500; Abcam). The remaining 360 μL of supernatant was added to 2 μL of anti-Flag antibody and incubated at 4°C for 4‒6 h. Then, 40–60 μL of protein A/G agarose was added and incubated at 4°C for 8–10 h. After centrifugation at 4°C and 1600
g for 3 min, supernatant was discarded, and the beads were mixed with 2× loading buffer and subject to western blot analysis using anti-ZEB1 antibody (ab203829, 1:500; Abcam).
4
Western Blot Analysis of EMT Markers
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In Western Blot analysis, cells were lysed in lyse buffer consisting of cell lytic M buffer (Sigma, St. Louis, USA) supplemented with 0.1% phosphatase-inhibitor (Sigma, St. Louis, MO, USA) and 0.1% protease-inhibitor (Sigma, St. Louis, MO, USA ). Isolated proteins (40 µg) were fractioned using 12% SDS gels and electro-transferred to a polyvinylidene difluoride membrane (Merck Millipore, Cork, Ireland). Primary antibodies against CTGF 1:1000 (#NB100-724, Novus Biologicals), RhoA 1:500 (#ARH04, Cytoskeleton, Denver, CO, USA), E-cadherin 1:1000 (CDH1; #ab40772, Abcam, Cambridge, Great Britain), SNAI2 1:500 (#ab180714, Abcam), vimentin 1:1000 (VIM; #ab92547, Abcam), ZEB1 1:500 (#ab203829, Abcam), and GAPDH 1:2000 (#5174S, Cell Signaling, Danvers, MA, USA) were used. Membrane was washed and incubated in horseradish peroxidase-conjugated secondary antibody (GE Healthcare, Buckinghamshire, UK). Antibody-bond protein bands were assayed using a chemiluminescent luminol enhancer solution (Cyanagen, Bologna, Italy) and detected by a C-DiGit Blot Scanner (LI-COR Bioscience, Lincoln, NE, USA). Full-length Western blot images are shown in the supplement.
5
Evaluating EMT Markers in Lung Cancer
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Human lung cancer cells were transfected with the relevant plasmids and cultured for 36 h. For western blot analysis, cells were lysed in NP-40 buffer (10 mM Tris pH 7.4, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA pH 8.0, 1 mM EGTA pH 8.0, 1 mM PMSF, and 0.5% NP-40) at 25 ˚C for 40 min. The lysates were added to 5× loading dye and then separated by electrophoresis. The primary antibodies used in this study were 1:1000 rabbit anti-Flag (sc-166384, Santa Cruz, Dallas, TX, USA) and 1:1000 Abcam (Cambridge, UK) antibody of UBE2C (ab12290), ZEB1 (ab203829), ZEB2 (ab138222), Vimentin (ab45939), E-cadherin (ab1416), cleaved Capase-3 (ab32042) and Tubulin (ab6046).
6
Western Blot Analysis of ZEB1, GAPDH, and β-actin
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Thirty micrograms of proteins were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to the nitrocellulose membrane. The membrane was blocked with 5% non-fat milk and incubated with the primary antibody for 1.5 hours. The protein band, specifically bound to the primary antibody, was detected using an IRDye 800CW-conjugated secondary antibody and LI-COR imaging system (LI-COR Biosciences). The primary antibodies were ZEB1 (1:1000; ab203829, Abcam), GAPDH (1:5000; ab181602, Abcam) and β-actin (1:5000; 60008-1-Ig, Proteintech).
7
Quantifying ZEB1 Expression in Mice Tumors
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IHC staining and score calculation were conducted as described previously (20 (link)). Anti-ZEB1 antibody (1:100; cat. no. ab203829; Abcam) was used to detect their expression levels in mouse tumors. Images were visualized using a Nikon ECLIPSE Ti (Nikon Corporation) inverted microscope system (x20 magnification) and processed using Nikon software (CaptureNX2, version 2.4.7).
8
Western Blot Analysis of EMT Markers
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Cells at the logarithmic growth phase were collected and lysed with radio immunoprecipitation assay lysis buffer (SS0501; SORFA, Beijing, China) to extract the total protein. The protein concentration was quantified using BCA kits (PC0020-500, Acmec, Shanghai, China). The protein samples were denatured by boiling, separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electrophoretically transferred onto the polyvinylidene fluoride membranes, and blocked with 5% skim milk for 1 h. Next, the membranes were incubated with rabbit anti-human primary antibodies anti-E-cadherin (1:10000, ab40772; Abcam, Cambridge, MA, USA), anti-N-cadherin (1:1000, ab207608; Abcam), anti-ZEB1 (1:1000, ab203829; Abcam), and β-actin (1:1000, ab8227, Abcam) at 4 °C overnight. After washing, the membranes were probed with HRP-labeled goat anti-rabbit secondary antibody IgG H&L (1:300, ab7090; Abcam) for 1 h. The membranes were visualized with enhanced chemiluminescence (ECL) and photographed. The gray value of the protein bands was analyzed using the Image-Pro 6.0 software with β-actin as the internal control.
9
Western Blot Analysis of EMT Markers
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The primary antibodies, including anti-Snail1 (ab216347, Abcam, Cambridge, MA, USA), anti-Zeb1 (ab203829, Abcam), anti-E-cadherin (ab40772, Abcam), anti-fibronectin (ab45688, Abcam), and anti-actin (ab179467, Abcam), were used for Western blot analysis. The Western blot analysis was performed as previously described [5 (link), 6 (link), 17 (link), 18 (link)].
10
Western Blot Analysis of Epithelial-Mesenchymal Transition Markers
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Cells were lysed with Radio-Immunoprecipitation Assay (RIPA) protein extraction reagent containing 50 mM Tris, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), sodium orthovanadate, sodium fluoride, ethylenediaminetetraacetic acid, and protease inhibitors (Beyotime, Beijing, China). The cell lysates were loaded on 10% SDS-polyacrylamide gel electrophoresis gel and transferred onto nitrocellulose membranes. The membranes were blocked with 5% nonfat milk and incubated with primary antibodies, anti-p21 antibody (1:1000, ab109520), anti-SNAIL antibody (1:500, ab180714), anti-ZEB1 antibody (1:1000, ab203829), and anti-GAPDH antibody (1:10 000, ab128915; Abcam, Cambridge, Massachusetts) at 4°C overnight. The membranes were then incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG H&L horseradish peroxidase (HRP; 1/5000, ab97051) or anti-rabbit HRP (1/2000, ab6721; Abcam, Cambridge, Massachusetts) secondary antibodies at room temperature for 1 hour, followed by detecting the visualized immune complexes with an enhanced chemiluminescence detection kit (Supersignal West Pico Trial kit, Pierce, lL, USA).
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