We used RIPA lysis buffer (Beyotime, China) to lyse cells and tissues. The concentrations of total protein were detected with a BCA Protein Assay Kit (TaKaRa). SDS-PAGE gels (10%) were used to separate proteins, and after electrophoresis, proteins were transferred to PVDF membranes. Then, membranes were blocked with 5% BSA. After blocking, membranes were incubated with rabbit polyclonal anti-human BTBD7 (ab154685; Abcam, 1 : 1000 dilution), rabbit polyclonal anti-human E-cadherin (ab133597; Abcam, 1 : 4000 dilution), rabbit polyclonal anti-human N-cadherin (ab76057; Abcam, 1 : 1000 dilution), rabbit polyclonal anti-human FN (ab76057; Abcam, 1 : 1000 dilution), and rabbit polyclonal anti-human vimentin (ab137321; Abcam, 1 : 2000 dilution) antibodies and with anti-β-actin (ab8227, Abcam, 1 : 3000 dilution) as the loading control. Each membrane was subsequently incubated with the corresponding secondary antibodies. Finally, we used an enhanced chemiluminescence (ECL) kit to visualize protein bands in a ChemiDoc XRS Plus luminescence image analyzer (Bio-Rad). The results are shown as relative grayscale values.
Ab76057
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab76057 is a primary antibody targeting a specific protein. It is designed for use in various laboratory applications, such as immunohistochemistry, Western blotting, and enzyme-linked immunosorbent assay (ELISA). The core function of this product is to detect and bind to the target protein, enabling researchers to study its expression and characteristics.
Automatically generated - may contain errors
Lab products found in correlation
Ab15148, by Abcam (17 mentions)
Ab40772, by Abcam (14 mentions)
Ab137321, by Abcam (12 mentions)
Pvdf membrane, by Merck Group (12 mentions)
Ab9485, by Abcam (11 mentions)
Bca kit, by Thermo Fisher Scientific (9 mentions)
Ab92547, by Abcam (8 mentions)
Ab181602, by Abcam (8 mentions)
Ab8978, by Abcam (8 mentions)
Ab1416, by Abcam (6 mentions)
Ab6721, by Abcam (6 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Ab16051, by Abcam (5 mentions)
Ab231303, by Abcam (5 mentions)
Ab8245, by Abcam (4 mentions)
Bca protein assay kit, by Beyotime (4 mentions)
Ab32503, by Abcam (4 mentions)
Ab76055, by Abcam (4 mentions)
Ripa buffer, by Beyotime (3 mentions)
Ab205718, by Abcam (3 mentions)
61 protocols using ab76057
1
Protein Expression Analysis of EMT Markers
2
Western Blot Analysis of Epithelial-Mesenchymal Transition Markers
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Samples were lysed with RIPA containing 1% protease and phosphate inhibitor cocktail (P8340; Sigma-Aldrich). Proteins were run on 10% SDS-PAGE and then transferred to PVDF membranes, which were incubated with primary antibodies at 4°C overnight and probed by secondary antibodies. Blots were visualized with enhanced chemiluminescence (WBKLS0500; Millipore, Temecula, CA) and the ChemiDoc Touch detection system (Bio-Rad). The following primary antibodies were used: slug (9585; Cell Signaling Technology, Danvers, MA; 1:1000), β-catenin (8480; Cell Signaling Technology; 1:1000), p-eIF2α (3398; Cell Signaling Technology; 1:1000), p-GSK3β (5558; Cell Signaling Technology; 1:1000), GAPDH (ab8245; Abcam, Cambridge, England; 1:1000), C/EBPβ (ab32358; Abcam; 1:1000), N-cadherin (ab76057; Abcam; 1:1000), MMP-2 (ab92536; Abcam; 1:1000), AKT (ab8805; Abcam; 1:1000), p-AKT (ab81283; Abcam; 1:1000), vimentin (ab92547; Abcam; 1:5000), and cleaved caspase-3 (19677-1-AP; Proteintech, Rosemont, IL; 1:500).
3
Immunofluorescence Staining of Cell Sections
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The samples were from cell sections and fixed with 4% formaldehyde for 30 min, rinsed three times with PBS, and permeabilized with PBST (0.1% Triton X-100 in PBS) for 5 min. After blocking with 3% BSA for 1 h and washing three times, the samples were incubated overnight at 4 °C; anti-MSX1 antibody (1:100, ab168745, Abcam, Cambridge, UK), anti-CDH2 antibody (1:100, ab76057, Abcam, Cambridge, UK), and anti-SPP1 antibody (1:150, WL02378, Wanlei Biotech, Shenyang, China) were used. After washing three times with PBS, incubation with Alexa-labeled donkey anti-mouse IgG (1:500, A16016, Invitrogen, Waltham MA, USA,) and Alexa-labeled donkey anti-rabbit IgG (1:500, A16028, Invitrogen, Waltham MA, USA) was performed at room temperature for 1 h. The nuclei were counterstained with 4’,6-diamidino-2-phenylindole for 5 min, and the cells were observed using laser-scanning confocal microscopy (Nikon, Melville, NY, USA). The negative control refers to incubating only with the Alexa-labeled secondary antibody, which are shown in Figure S4 .
4
Western Blot Analysis of EMT Markers
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Transfected HCCLM3 and PLC5 cells were lysed utilizing RIPA buffer (Beyotime, Shanghai, China). The quality of protein samples was assessed via BCA method. Then, the samples were separated by SDS–PAGE, followed by being transferred to PVDF membranes. The membranes were blocked with 5% skimmed milk powder blocking solution at room temperature for 2 h. Afterwards, the membranes were incubated with primary antibodies against E-cadherin (1:10000; ab40772, Abcam, USA) N-cadherin (1:1000; ab76057, Abcam), vimentin (1:1000; 741, Cell Signaling Technology, USA), BICD2 (1:1000; ab237616, Abcam), and GAPDH (1:1000; ab181602, Abcam) overnight at 4°C, followed by being incubated with HRP-conjugated secondary antibodies (1:1000; ab7097, Abcam) at room temperature lasting 2 h. Enhanced chemiluminescence kit (Beyotime) was used for visualizing the protein bands. The grayscale density was measured by ImageJ software.
5
Immunohistochemical Analysis of Xenograft Tumors
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Paraffin sections of xenograft tumor tissues were cut with a thickness of 4 μm. Sections were incubated with primary antibodies: anti-RTKN2 (Abcam, #ab251807, 1:1000), anti-β-catenin (Abcam, #ab16051, 1:1000), anti-Ki67 (Abcam, #ab245113, 1:1000), anti-E-cadherin (Abcam, #ab1416, 1:500), and anti-N-cadherin (Abcam, #ab76057, 1:1000). The temperature was maintained at 4 °C overnight. Sections were co-incubated with HRP‐polymer‐conjugated secondary antibodies after washing with phosphate‐buffered saline, then, they were immunostained using DAB plus kit.
6
Immunohistochemical Analysis of EMT Markers
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Paraffin sections were baked at 56 °C for 2 h for dewaxing, boiled in citrate buffer for antigen retrieval, and blocked using 3% hydrogen peroxide. Sections were probed overnight at 4°C with anti-FEN1 (A1175, Abclonal, China), anti-E-cadherin (ab40772, Abcam, Cambridge, UK), anti-N-cadherin (ab76057, Abcam, Cambridge, UK), and anti-Vimentin (ab92547, Abcam, Cambridge, UK) antibodies, followed by incubation with HRP-conjugated secondary antibodies for 1 h at 37 °C. Sections were stained using DAB coloration kits and counterstained with hematoxylin for 10 s. Samples were dehydrated using a graded series of ethanol. Image-Pro Plus software was used to measure IHC staining intensity. IHC scores were calculated by multiplying the percentage score and intensity score of stained cells. The percentage of positively stained cells was scored on a scale from 0 to 100. Staining intensity (0 = negative staining; 1 = weak; 2 = moderate; 3 = strong) was scored on a scale from 0 to 3. Thus, the IHC score ranged from 0 to 300. The mean IHC score was defined as the cut-off value for low and high expression.
7
Protein Expression Analysis of Cancer Markers
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The total proteins were isolated by using lysis buffer containing a protease inhibitor cocktail (P8340; Sigma). The protein concentration in each sample was evaluated by using a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). A 30 µg sample of total protein from each sample was separated by 10% SDS-PAGE, and the separated protein bands were transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA), which were then blocked with skimmed milk (5%) for 2 hrs at room temperature. Next, the membranes were incubated overnight at 4 °C with primary antibodies, followed by incubation with the secondary antibodies (HRP linked, Abcam, Cambridge, UK, ab205718, 1:2,000) at room temperature for 2 hrs. The blots were exposed by using ECL detection reagent (EMD Millipore) and analyzed by using ImageJ version 1.48 software (National Institutes of Health, Bethesda, MD, USA). The primary antibodies used in this study were anti-E-cadherin (Abcam, ab76319, 1:1,000), anti-MMP2 (Abcam, ab2462, 1:1,000), anti-MMP9 (Abcam, ab228402, 1:1,000), anti-N-cadherin (Abcam, ab76057, 1:1,000), anti-Bax (Abcam, ab53154, 1:1,000), anti-Caspase-3 (Abcam, ab214430, 1:1,000), and anti-GAPDH (Abcam, ab9485, 1:2,000).
8
Immunofluorescence Staining of Adherent Cells
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Cells were plated at 20,000 cells/well on coverslips in 24 well plates and allowed to attach overnight. Wells were fixed, permeabilized, and blocked as described previously [54 ]. Cells stained on cover slips were mounted on slides using Fluorescence Mounting Medium (S3023, DAKO). The coverslips were then incubated for 16 hours at 4°C with polyclonal rabbit anti-CK19 (10 μg/ml, ab15463, Abcam) or polyclonal rabbit anti-N-cadherin (1:100, ab76057, Abcam). Alexa Fluor 594 polyclonal goat anti-rabbit (1:700, A-11012, Life Technologies) was used as a secondary antibody in 1% BSA (A2153, Sigma-Aldrich) in PBS for 1 hour at room temperature. For L1CAM, monoclonal anti-L1CAM-PE (1:50, ab95694, Abcam) was used. Nuclei were counterstained with DAPI (1 μg/ml, 10236276001, Roche,), in PBS for 5 minutes at room temperature. Images were obtained using an Axiovert 200M fluorescence microscope (Zeiss Axiovert 200 M, Carl Zeiss MicroImaging GmbH, Jena, Germany) with CCD camera (Zeiss Axiocam HR), using Axiovision software. All images are taken with 40x magnification.
9
Protein Expression Analysis in Cancer Cells
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LoVo or SW1116 cells were lysed on ice with the lysis buffer (Beyotime, Shanghai, China) and the lysates were later centrifuged for 15 min at 12,000 × g. The BCA kit (Thermo Fisher Scientific, Waltham, MA, USA) was acquired for evaluating protein concentrations. Protein was subjected to SDS–PAGE (Bio-Rad, Hercules, CA, USA) and transferred to PVDF membranes (Millipore, Billerica, MA, USA). Primary antibodies and secondary antibodies were applied to incubate membranes in sequence. ECL detection system (Applied Biosystems, Foster City, CA, USA) was utilized to visualize protein bands. Primary antibodies against MMP2 (ab97779, Abcam, Cambridge, MA, USA), MMP7 (ab5706, Abcam), N-cadherin (ab76057, Abcam), E-cadherin (ab40772, Abcam), NANOG (ab80892, Abcam), OCT4 (ab181557, Abcam), Gli4 (AV37797, Sigma-Aldrich), PTCH1 (ab53715, Abcam), Shh (ab53281, Abcam), Gli1 (ab49314, Abcam), Gli2 (ab167389, Abcam), and GAPDH (ab9484, Abcam) were used, individually.
10
Immunohistochemical Analysis of Lung Metastasis
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Tissue sections were treated with Ki67 (1:500, ab15580, Abcam), N-cadherin (1:1000, ab76057, Abcam) and E-cadherin (1:500, ab15148, Abcam). The percentage of Ki67-positive cells was quantified by Image ProPlus 6.0. The lung sections of nude mice were dewaxed with xylene, washed with distilled water after hydration, and the metastasis was observed after hematoxylin and eosin (HE) staining.
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