Total protein was extracted from cells 72 h after transfection or 48 h after different doses irradiation using RIPA lysis buffer (Beyotime Institute of Biotechnology). Protein concentrations were determined using an enhanced BCA protein assay kit (Beyotime Institute of Biotechnology), and a total of 20 μg protein was loaded onto 10% SDS-PAGE gels. The separated proteins were then transferred onto 0.2 µm PVDF membranes (Roche Applied Science). Following blocking with 5% skimmed milk for 1 h at room temperature, the membranes were incubated with primary antibodies overnight at 4 °C. The primary antibodies used in the present study included C1GALT1 (1:5000; ab237734; Abcam), E-cadherin (1:5000; ab40772; Abcam), N-cadherin (1:5000; ab76011; Abcam), vimentin (1:5000, ab92547; Abcam), and GADPH (1:5000, ab9485; Abcam). Subsequently, the membranes were incubated with anti-rabbit secondary antibody (1:20,000; 7074P2; Cell Signaling Technology) conjugated with horseradish peroxidase for 1 h at room temperature. Protein bands were detected using Pierce ECL reagents (Thermo Fisher Scientific, Inc.) and visualized with a Tanon-5200 imaging system (Tanon Science and Technology Co., Ltd.).
Ab237734
Manufactured by Abcam
Sourced in United States
Ab237734 is a lab equipment product offered by Abcam. It is designed for general laboratory use. The core function of this product is to support various experimental procedures. Further details on the specific intended use are not provided.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Beyotime (2 mentions)
Ab124804, by Abcam (2 mentions)
Ab40772, by Abcam (1 mentions)
Ab76011, by Abcam (1 mentions)
Pierce ecl reagent, by Thermo Fisher Scientific (1 mentions)
Ab92547, by Abcam (1 mentions)
Ab9485, by Abcam (1 mentions)
Pvdf membrane, by Roche (1 mentions)
Enhanced bca protein assay kit, by Beyotime (1 mentions)
Ab150361, by Abcam (1 mentions)
Rac1 activation assay kit, by Cytoskeleton (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ab52971, by Abcam (1 mentions)
Bl006b, by Biosharp (1 mentions)
Transscript first strand cdna synthesis supermix, by Transgene (1 mentions)
Bca protein assay kit, by Biosharp (1 mentions)
Ab150179, by Abcam (1 mentions)
Ab150118, by Abcam (1 mentions)
A11008, by Thermo Fisher Scientific (1 mentions)
Fluoromount, by Diagnostic BioSystems (1 mentions)
5 protocols using ab237734
1
Western Blot Analysis of Protein Expression
2
Quantitative Immunohistochemical Analysis
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IHC was conducted as previously described [34 (link)]. The staining degree was calculated as follows: Overall score = intensity score (0, negative; 1, weak; 2, moderate; and 3, strong) × percentage score (0, 0–5%; 1, 6–25%; 2, 26–50%; 3, 51–75%; 4, ≥ 76%). A final score ≥ 4 was considered as high expression and < 4 as low expression. The following antibodies were used: C1GALT1 (ab237734, 1:500, Abcam, USA), integrin α5 (ab150361, 1:100, Abcam), p-PI3K (abs103557, 1:200, Absin, China), p-AKT (abs130889, 1:150, Absin), and SP1 (ab124804, 1:150, Abcam).
3
Protein Extraction and Western Blot Analysis
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Total protein was extracted using RIPA lysis buffer (Beyotime, Shanghai, China). After quantification, an equal amount of protein was run on a 10% SDS-PAGE gel and transferred to a PVDF membrane (Millipore, Billerica, MA, United States). After blocking with 5% BSA, the membrane was probed with the indicated antibodies. Primary antibodies used in this study were as follows: C1GALT1 (Abcam, Cambridge, United Kingdom, ab237734), RAC1 (ab155938), and GADPH (ab9485). Reactive bands were visualized using an ECL system (Pierce, Rockford, IL, United States). RAC1 activity was determined using the Rac1 activation assay kit (Cytoskeleton, Denver, CO, United States). GTP-RAC1 was detected by Western blot using an anti-RAC1 antibody.
4
Quantitative Analysis of Gene and Protein Expression
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Total RNA was reverse-transcribed into cDNA using TransScript First-Strand cDNA Synthesis SuperMix (TransGen, China). qRT-PCR reactions were conducted using SYBR PCR Master Mix (ABI, USA). The primers were listed in Additional file 1 : Table S3. The relative gene expression was normalized to control using the 2−ΔΔCt method. Total protein was quantified using the BCA protein assay kit (Biosharp, China). The approach for Western blot was conducted as described previously [34 (link)]. The following antibodies were used: C1GALT1 (ab237734, Abcam), integrin α5 (ab52971, Abcam), PI3K (4255, CST, USA), p-PI3K (4228, CST), AKT (9272, CST), p-AKT (9271, CST), SP1 (ab124804, Abcam), and GADPH (BL006B, Biosharp).
5
Kidney Tissue Immunofluorescence Analysis
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The human kidney tissues were embedded in the OCT compound, and 4-μm-thick serial cryostat sections were prepared. They were blocked for 30 min with 3% bovine serum albumin and stained overnight at 4°C with several lectins or antibodies: FITCconjugated Amaranthus caudatus lectin (ACA: F-8021-2; EY Laboratories), FITC-conjugated Agaricus bisporus lectin (ABA: F-5001-1; EY Laboratories), FITC-conjugated Maackia amurensis lectin (MAH: F-7801-2; EY Laboratories), anti-C1GALT1 rabbit polyclonal antibody (ab237734; Abcam), anti-COSMC rabbit polyclonal antibody (ab229831; Abcam), anti-Aquaporin 1 mouse monoclonal antibody (ab9566), and anti-Uromodulin sheep polyclonal antibody (PA5-46959; Thermo Fisher Scientific). The sections were then incubated at room temperature for 1 h with Alexa Fluor 488-conjugated anti-rabbit antibody (goat polyclonal antibody, A11008; Thermo Fisher Scientific), Alexa Fluor 555-conjugated anti-mouse antibody (goat polyclonal antibody, ab150118; Abcam), Alexa Fluor 647-conjugated anti-sheep antibody (donkey polyclonal antibody, ab150179; Abcam), and with DAPI (Sigma-Aldrich) for nuclear staining. The sections were then mounted with Fluoromount (Diagnostic BioSystems). Representative images were acquired with the all-in-one Fluorescence Microscope BZ-X700 (Keyence).
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