After different proportions of MSC and MG63 were co-cultured to a predetermined time, the supernatant of the upper and lower chambers of the 6-well cell culture plate was aspirated, and cells were cleaned twice with PBS. A protein extraction kit (SD-001, Invent Biotechnologies, Inc, China) was used for protein extraction. Lysis buffer was added and spilled on the ice for 5 min. Then, the protein lysate was filtered through a centrifugal column and placed into a centrifuge for 10 minutes under the condition of 10000 rpm and 4°C, and the clear fluid in the collection tube is the protein. NanoDrop 2000 was used to measure protein concentration. After that, the protein was prepared with a 5x loading buffer in proportion, and the sample was performed using publishing protocols [16 (link), 17 (link)] with antibodies directed against α-smooth muscle actin (α-SMA, ab5694, Abcam, Cambridge, UK), E-cadherin (ab212059, Abcam), N-cadherin (AF4039, Affinity), Slug (ab51772, Abcam), Snail (ab216347, Abcam), and Vimentin (ab92547, Abcam); the dilution of these antibodies was 1 : 1000, and β-actin (ab8227, Abcam) and GAPDH (ab9485, Abcam) ratio of these two antibodies was 1 : 100000.
Ab212059
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab212059 is a primary antibody that specifically binds to the target antigen. The core function of this product is to facilitate the detection and identification of the target protein in various experimental applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab76011, by Abcam (5 mentions)
Ab8227, by Abcam (4 mentions)
Ab216347, by Abcam (3 mentions)
Ab205718, by Abcam (3 mentions)
Ab92547, by Abcam (2 mentions)
Bicinchoninic acid (bca), by Beyotime (2 mentions)
Ab223075, by Abcam (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Chemiluminescence system, by Bio-Rad (2 mentions)
Radioimmunoprecipitation buffer, by Beyotime (2 mentions)
Ab51772, by Abcam (1 mentions)
Sd 001, by Invent Biotechnologies (1 mentions)
Af4039, by Affinity Biosciences (1 mentions)
Ab9485, by Abcam (1 mentions)
Ab5694, by Abcam (1 mentions)
Ab15251, by Abcam (1 mentions)
Hypersensitive ecl kit, by Beyotime (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Ab150077, by Abcam (1 mentions)
9 protocols using ab212059
1
Protein Expression Analysis of MSC-MG63 Co-culture
2
Western Blot Analysis of Cell Signaling Proteins
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The GC cells were lysed employing RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) containing protease inhibitors. After high-speed centrifugation, the supernatant was collected and heated in a 100°C water bath for 10 min to denature the protein. The proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) after protein quantification by the BCA protein assay kit (Beyotime Biotechnology, Shanghai, China), and then transferred onto the PVDF membrane. Then, the membranes were washed with tris buffered saline tween (TBST) solution, and they were incubated overnight with rabbit anti-N-cadherin antibody (Abcam, ab76011, 1:500), rabbit anti-E-cadherin antibody (Abcam, ab212059, 1:500), rabbit anti-Wnt1 antibody (Abcam, ab15251, 1:500), rabbit anti-β-catenin (Abcam, ab32572, 1:500), rabbit anti-β-actin antibody (Abcam, ab8227, 1:500) at 4°C. After rinsing the PVDF membranes with TBST solution again, they were incubated with goat anti-rabbit IgG H&L (HRP) (Abcam, ab150077, 1:1000) at room temperature for 1 h. After rinsing the membranes with TBST again, the protein bands were developed by the hyper-sensitive ECL kit (Beyotime Biotechnology, Shanghai, China).
3
Extracellular Vesicle Protein Characterization
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Following total protein extraction from cells or tissues, protein concentration was determined using a BCA assay kit (Thermo Fisher Scientific). Then, 30 μg of total protein was subjected to PAGE, transferred onto a PVDF membrane (Amersham, Little Chalfont, UK) and sealed. Thereafter, the membrane was probed with primary rabbit antibodies overnight at 4°C and then with HRP-labeled secondary antibody goat anti-rabbit (1: 5000, ab6721, Abcam). The membrane was developed with an optical luminometer (GE Healthcare, Little Chalfont, Buckinghamshire, UK), and quantified using Image Pro Plus 6.0 software. Primary antibodies used were as follows: CD63 (1: 1000, ab134045, Abcam, Cambridge, UK), Alix (1 μg/mL, ab76608, Abcam), TSG101 (1: 1000, ab125011, Abcam), Calnexin (1: 1000, ab22595, Abcam), β-actin (1: 5000, ab8227, Abcam), INHBA (1: 1000, ab128958, Abcam), IL13RA2 (1: 1000, ab55275, Abcam), E-cadherin (1: 1000, ab212059, Abcam), N-cadherin (1: 1000, ab19348, Abcam), Vimentin (1: 1000, ab45939, Abcam).
4
Protein Expression Analysis via Western Blot
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After extracting by RIPA lysis buffer (Solarbio), protein was electrophoresed by SDS‐PAGE gel and then transferred to the PVDF membrane (Merck). Then, the membrane was incubated with the antibodies against HK2 (ab209847; 1:1000; Abcam), N‐cadherin (N‐cad, ABP51903; 1:1000; Abbkine), E‐cadherin (E‐cad, ab212059; 1:1000; Abcam), Lactate Dehydrogenase (LDHA, ab134187; 1:500; Abcam), Glucose Transporter (GLUT1, ab115730; 1:1000; Abcam), and β‐actin (ABP50593; 1:2000; Abbkine) overnight at 4°C. Then, membrane was hatched with secondary antibody (ab205718; 1:5000; Abcam) for 2 h. All membranes were incubated with ECL reagent (Abcam) and photographed in a gel imaging analyzer (UVITEC).
5
Protein Expression Analysis Protocol
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The proteins were measured with BCA method. SDS-PAGE was conducted, and protein samples were transferred to a PVDF membrane (Milipore, GVWP02500, US). The membrane was blocked with non-fat milk, and washed with TBST. Then, the membranes were incubated with primary antibodies and second antibodies, successively. Finally, enhanced chemiluminescence detection kit (Thermo Fisher, USA) was used in immunoblots. Rabbit monoclonal to Moesin (1:1000, ab169789, Abcam, UK), rabbit monoclonal to N-Cadherin (1:1000, ab76011), rabbit monoclonal to E-Cadherin (1:2000, ab212059), rabbit polyclonal to beta-actin (1:3000, ab8227).
6
Dissecting the Role of NCAPH in EMT and PI3K/AKT Signaling
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NCAPH antibody (1:500 dilution, ab154105, Abcam), E-cadherin antibody (1:500 dilution, ab212059, Abcam), N-cadherin antibody (1:1000 dilution, ab76011, Abcam), Vimentin (1:500 dilution, ab92547, Abcam), ZO-1 antibody (1:500 dilution, ab221547, Abcam), p-PI3K antibody (1:1000 dilution, ab278691, Abcam), PI3K antibody (1:500 dilution, ab32089, Abcam), p-AKT antibody (1:500 dilution, ab38449, Abcam), AKT antibody (1:500 dilution, ab8805, Abcam), and beta-actin antibody (1:2000 dilution, ab8226, Abcam) were obtained from the indicated company.
Physiology International 109 (2022) 3, 334-347
The NC (negative control) siRNAs and two NCAPH siRNAs were bought from Riobio. The sequence of NCAPH siRNA was #1: 5 0 -GAGUUCAGGAGCUGGAAGG-3 0 . #2: 5 0 -ACCA-CAGGGAAGCUGGAAA-3 0 .
The plasmids including pcDNA3.1-vector and pcDNA3.1-control were constructed in our lab. LY294002 was bought from Sigma.
Physiology International 109 (2022) 3, 334-347
The NC (negative control) siRNAs and two NCAPH siRNAs were bought from Riobio. The sequence of NCAPH siRNA was #1: 5 0 -GAGUUCAGGAGCUGGAAGG-3 0 . #2: 5 0 -ACCA-CAGGGAAGCUGGAAA-3 0 .
The plasmids including pcDNA3.1-vector and pcDNA3.1-control were constructed in our lab. LY294002 was bought from Sigma.
7
Western Blot Analysis of EMT Markers
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Radioimmunoprecipitation buffer (Beyotime, Beijing, China) was used for lysing cellson ice within 30 min. The bicinchoninic acid (Beyotime, Beijing, China) was used for quantifying the protein concentrations. Lysates were resolved by SDS-PAGE and transferred onto polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). The membranes were blocked and incubated with primary antibodies against N-cadherin (ab76011, 1:1,000, Abcam, Cambridge, MA, USA), E-cadherin (ab212059, 1:1,000, Abcam, Cambridge, MA, USA), β-catenin (ab223075, 1:1,000, Abcam, Cambridge, MA, USA) and snail (ab216347, 1:1,000, Abcam, Cambridge, MA, USA) overnight at 4 °C and then incubated with HRP-conjugated secondary monoclonal antibody (ab205718, 1:5,000, Abcam, Cambridge, MA, USA) for 1 h at room temperature. The chemiluminescence system (Bio-Rad) was used for quantifying the protein of the gels.
8
Immunohistochemical Analysis of Tissue Markers
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Paraffin-embedded blocks were cut into 4-μm-thick sections, and the sections were dewaxed and hydrated. Then, the sections were immersed in distilled water containing 3% hydrogen peroxidase twice to reduce endogenous oxidase activity. Next, the tissue sections were incubated with anti-FSP1 antibody (Abcam, ab197896), anti-α-SMA antibody (Abcam, ab124964), anti-ZO-1 antibody (Abcam, ab221546) and anti-E-cadherin antibody (Abcam, ab212059) for 2 h at room temperature, and subsequently, a goat-anti-rabbit antibody was applied to the cells at room temperature for 40 min. The degree of staining was determined by developing with diaminobenzidine (DAB) chromogen (Bio-Rad, Inc., CA, United States). Subsequently, the tissue sections were dehydrated and sealed with gum. Five random fields of view at 100× magnification were selected with a camera using a microscope (Olympus, Japan), and the mean microvessel count was recorded as the microvessel density.
9
Analyzing Epithelial-Mesenchymal Transition Markers
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Cells were lysed with radioimmunoprecipitation buffer (Beyotime, Beijing, China) on ice for 30 min. The protein concentrations were quantified using bicinchoninic acid (Beyotime, Beijing, China). Lysates were resolved by SDS-PAGE and transferred onto polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). The membranes were blocked and incubated with primary antibodies against E-cadherin (ab212059, 1:1,000, Abcam, Cambridge, MA, USA), N-cadherin (ab76011, 1:1,000, Abcam, Cambridge, MA, USA), snail (ab216347, 1:1,000, Abcam, Cambridge, MA, USA) and β-catenin (ab223075, 1:1,000, Abcam, Cambridge, MA, USA) overnight at 4 °C and then incubated with HRP-conjugated secondary monoclonal antibody (ab205718, 1:5,000, Abcam, Cambridge, MA, USA) for 1 h at room temperature. The gels were detected using a chemiluminescence system (Bio-Rad) and the protein was quantified.
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