After the experiment, the colon tissues of mice were collected, and the paraffin-embedded mouse colon tissues (thickness of 4 µm) were fixed with formalin. Following deparaffinization and hydration, the sections were treated with 3% hydrogen peroxide to block endogenous peroxidase activity, and antigenic repair was achieved by boiling the sections in citrate buffer for 30 min. Next, the sections were sealed with 5% bovine serum albumin (BSA) and incubated overnight at 4°C with primary antibodies PCNA (AB29, dilution ratio of 1: 10,000, Abcam, Cambridge, United Kingdom), VEGFA (AB133352, dilution ratio of 1: 250, Abcam), PI3K (95,625, dilution ratio of 1: 400, CST, Boston, Massachusetts), and Akt (95,625, 1: 400, CST). Afterward, the sections were stained with diaminobenzidine (DAB, Boster Biological Technology, Pleasanton, CA) substrate, and the nuclei were stained with hematoxylin.
Ab133352
Manufactured by Abcam
Sourced in United Kingdom
Ab133352 is a recombinant protein product. It is produced using a baculovirus expression system.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Merck Group (2 mentions)
Ab11934, by Abcam (2 mentions)
Ab16066, by Abcam (2 mentions)
Ab180158, by Abcam (2 mentions)
Ab190085, by Abcam (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Ab216327, by Abcam (2 mentions)
Vegfa, by Abcam (1 mentions)
Diaminobenzidine dab, by Boster Bio (1 mentions)
Ecl detection system, by Merck Group (1 mentions)
Ab2787, by Abcam (1 mentions)
Ab81371, by Abcam (1 mentions)
Ab51608, by Abcam (1 mentions)
Bicinchoninic acid assay, by Thermo Fisher Scientific (1 mentions)
Ecl plus substrate, by Thermo Fisher Scientific (1 mentions)
Ab92494, by Abcam (1 mentions)
Ab3742, by Abcam (1 mentions)
Ab181557, by Abcam (1 mentions)
Ab75854, by Abcam (1 mentions)
Ab184787, by Abcam (1 mentions)
8 protocols using ab133352
1
Immunohistochemical Analysis of Colon Tissues
2
Protein Expression and Quantification
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Total cell protein was extracted. The protein concentration was determined by BCA method. SDS polyacrylamide gel electrophoresis was performed on 60 μg total proteins in each group. Semidry transfer membrane, lichun red staining marks. 100 g/L skimmed milk powder was sealed at room temperature for 2 h. Diluted primary antibody, anti-Hsp70 antibody (ab2787, 1 : 1000 dilution; Abcam, Cambridge, MA, USA), anti-HIF-1 alpha antibody (ab51608, 1 : 1000 dilution), anti-Sumo 1 antibody (ab133352, 1 : 1000 dilution), anti-Sumo 2 + Sumo 3 antibody (ab81371, 1 : 1000 dilution) with 50 g/L skimmed milk powder, then incubate at 4°C overnight. TBST was used to wash 3 times at room temperature, for 10–15 min. Diluted HRP-labeled II antibody (1 : 10,000, Thermo Fisher) with skimmed milk powder of 50 mL/L and then incubate at room temperature for 2 h, and TBST was used to wash 3 times at room temperature, for 10–15 min. Detection was carried out by ECL detection system (Millipore).
3
Western Blot Analysis of SUMO Pathway
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After extraction of protein from NSCs, the protein concentrations were determined by bicinchoninic acid assay (Thermo Fisher Scientific, Waltham, MA). Twenty micrograms protein of each sample in an equal volume was electrophoresed on 10%–12% polyacrylamide gels, before transfer onto a polyvinylidene fluoride membrane. After blocking the membrane in skim milk for 1 h, antibodies against SUMO1 (ab133352, 1 : 1000; Abcam, Cambridge, UK), SUMO2/3 (ab3742, 1 : 1000), UBC9 (ab75854, 1 : 1000), Oct4 (ab181557, 1 : 1000), SOX2 (ab92494, 1 : 1000), caspase-3 (ab184787, 1 : 1000), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; ab9485, 1 : 1000) were incubated overnight at 4°C. Then, the membranes were washed five times with TBST, followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG (111-035-003; 1 : 2,000; Jackson Immuno Research Laboratories, Inc., West Grove, PA, USA) antibody for 1 h at room temperature. GAPDH was used as internal control. Finally, membranes were exposed using ECL Plus substrate (Thermo Fisher Scientific, Waltham, MA). Data were evaluated by image analysis software (ImageJ version 1.48; National Institutes of Health, Bethesda, MD, USA).
4
PPARγ and SUMO1 Identification in Meibocytes
5
SUMO1 Regulation in Mouse Tissues
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SENP1 antibody-C12 (1:500, sc-271360; Santa Cruz Biotechnology), SUMO1 antibody (1:1,000, ab133352; Abcam), and β-actin antibody (1:2,000, sc-47778; Santa Cruz Biotechnology) were used as primary antibodies. Anti-mouse (1:5,000, NA934V; GE Healthcare) or anti-rabbit (1:5,000, NA931V; GE Healthcare) were used as secondary antibodies. Mouse islets, gut, and brain were homogenized with 7 mol/L guanidine HCl. The protein was precipitated by addition of methanol, chloroform, and water in a 4:1:3 (v/v) ratio, and the pellet was recovered (10,000 rpm for 10 min) and dissolved in 1% SDS, 0.2 mol/L Tris, 10 mmol/L dithiothreitol, pH 6.5. Protein concentration was estimated by absorbance at 280 nm. Fifty, 10, and 10 µg protein from islets, intestine, and brain, respectively, were separated by SDS-PAGE (7.5% gel), transferred to polyvinylidene fluoride membrane, and probed with primary antibody in the presence of 5% skim milk. For SUMOylation (19 (link)), islets were incubated in Krebs-Ringer bicarbonate HEPES buffer with 2.8 mmol/L glucose for 2 h followed by 16.7 mmol/L glucose for 15 min. Islets were washed with cold PBS and put in lysis buffer with 10 mmol/L N-ethylmaleimide (128-53-0; MilliporeSigma), 100 µL PhosStop (1 tablet in 1 mL lysis buffer; MilliporeSigma), and 10 μL protease inhibitor (P8340; MilliporeSigma). Ten micrograms of protein were loaded for SDS-PAGE (10% gel).
6
Western Blot Analysis of Tight Junction Proteins
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Whole protein lysates were extracted from different samples using RIPA lysis buffer (JRDUN, Shanghai, P.R. China) with EDTA-free Protease inhibitor Cocktail (Roche, Heidelberg, Germany). The concentration of protein samples was determined by an enhanced BCA protein assay kit (Thermo Fisher Scienti c).
Equal amounts of total protein (25 mg) were fractionated on 10% SDS-PAGE and transferred to a nitrocellulose membrane (Millipore, Billerica, MA, USA) overnight. Then, after being blocked with 5% nonfat dry milk for 1 hour at room temperature, the membranes were probed at 4°C overnight with the primary antibodies followed by secondary antibody anti-mouse IgG (1:1,000; Beyotime, Shanghai, P.R. China) for 1 hour at 37°C. An enhanced chemiluminescence system (Tanon, Shanghai, China) was used for detecting protein expression value. The information of primary antibodies is provided as follows: SUMO1 (ab133352, Abcam, UK), HIF-1α (ab16066, abcam, UK), VEGF (ab11934, Abcam, UK), occluding (ab216327, Abcam, UK), claudin-1(ab180158, Abcam, UK), ZO-1(ab190085, Abcam, UK) and GAPDH (#5174, CST, USA).
Equal amounts of total protein (25 mg) were fractionated on 10% SDS-PAGE and transferred to a nitrocellulose membrane (Millipore, Billerica, MA, USA) overnight. Then, after being blocked with 5% nonfat dry milk for 1 hour at room temperature, the membranes were probed at 4°C overnight with the primary antibodies followed by secondary antibody anti-mouse IgG (1:1,000; Beyotime, Shanghai, P.R. China) for 1 hour at 37°C. An enhanced chemiluminescence system (Tanon, Shanghai, China) was used for detecting protein expression value. The information of primary antibodies is provided as follows: SUMO1 (ab133352, Abcam, UK), HIF-1α (ab16066, abcam, UK), VEGF (ab11934, Abcam, UK), occluding (ab216327, Abcam, UK), claudin-1(ab180158, Abcam, UK), ZO-1(ab190085, Abcam, UK) and GAPDH (#5174, CST, USA).
7
Western Blot Analysis of Tight Junction Proteins
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Whole protein lysates were extracted from different samples using RIPA lysis buffer (JRDUN, Shanghai, P.R. China) with EDTA-free Protease inhibitor Cocktail (Roche, Heidelberg, Germany). The concentration of protein samples was determined by an enhanced BCA protein assay kit (Thermo Fisher Scienti c).
Equal amounts of total protein (25 mg) were fractionated on 10% SDS-PAGE and transferred to a nitrocellulose membrane (Millipore, Billerica, MA, USA) overnight. Then, after being blocked with 5% nonfat dry milk for 1 hour at room temperature, the membranes were probed at 4°C overnight with the primary antibodies followed by secondary antibody anti-mouse IgG (1:1,000; Beyotime, Shanghai, P.R. China) for 1 hour at 37°C. An enhanced chemiluminescence system (Tanon, Shanghai, China) was used for detecting protein expression value. The information of primary antibodies is provided as follows: SUMO1 (ab133352, Abcam, UK), HIF-1α (ab16066, abcam, UK), VEGF (ab11934, Abcam, UK), occluding (ab216327, Abcam, UK), claudin-1(ab180158, Abcam, UK), ZO-1(ab190085, Abcam, UK) and GAPDH (#5174, CST, USA).
Equal amounts of total protein (25 mg) were fractionated on 10% SDS-PAGE and transferred to a nitrocellulose membrane (Millipore, Billerica, MA, USA) overnight. Then, after being blocked with 5% nonfat dry milk for 1 hour at room temperature, the membranes were probed at 4°C overnight with the primary antibodies followed by secondary antibody anti-mouse IgG (1:1,000; Beyotime, Shanghai, P.R. China) for 1 hour at 37°C. An enhanced chemiluminescence system (Tanon, Shanghai, China) was used for detecting protein expression value. The information of primary antibodies is provided as follows: SUMO1 (ab133352, Abcam, UK), HIF-1α (ab16066, abcam, UK), VEGF (ab11934, Abcam, UK), occluding (ab216327, Abcam, UK), claudin-1(ab180158, Abcam, UK), ZO-1(ab190085, Abcam, UK) and GAPDH (#5174, CST, USA).
8
Quantifying SIRT1 and SUMO Pathway Proteins
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Human peripheral blood lymphocyte separation solution was purchased from Tianjin Haoyang Biological Manufacture Co., Ltd. (Tianjin, China). Radioimmunoprecipitation assay (RIPA) protein lysis buffer was obtained from Amyjet Scientific, Inc. (Wuhan, China). A BCA protein assay kit was purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). Antibodies used included: Rabbit anti-human SIRT1 antibody (Santa Cruz Biotechnology Inc., Dallas, TX, USA), rabbit anti-human GAPDH antibody (Beyotime Institute of Biotechnology, Inc., Jiangsu, China), horseradish peroxidase (HRP)-labeled goat anti-rabbit immunoglobulin G (IgG) antibody (Shanghai YuanMu Biological Technology Co., Ltd., Shanghai, China), anti-SUMO1 monoclonal antibody (ab133352; Abcam, Cambridge, UK), anti-SUMO2/3 polyclonal antibody (ab3742; EMD Millipore, Billerica, MA, USA), and anti-SUMO4 monoclonal antibody (ab126606; Abcam).
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