Protein samples were harvested from cell lysates and the concentration of the concentration of total protein was measured with a BCA Protein assay kit (Beyotime Biotechnology, Bejing, China). Equal amount of protein extracts was separated by 10% sodium dodecyl sulphate-polyacrylamide gels (SDS-PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes (Thermo Fisher Scientific). The membranes were blocked for 1 h in in PBS containing 5% nonfat milk at room temperature and then incubated with primary antibodies at 4 °C overnight: rabbit anti-TNFAIP8 antibody (1:500 dilution; ab166804, Abcam, MA, USA), rabbit anti-p53 antibody (1:500 dilution; ab131442), rabbit anti-E-cadherin antibody (1:500 dilution; ab15148), rabbit anti-N-cadherin antibody (1:500 dilution; ab18203), rabbit-anti-Vimentin antibody (1:1000 dilution; ab92547), rabbit anti-Snail antibody (1:500 dilution; ab82846) and rabbit anti-β-actin antibody (1:1000 dilution; #4970, Cell Signaling Technology, MA, USA). After being washed with PBS, the membrane was probed with corresponding goat anti-rabbit IgG H&L secondary antibodies for 1 h (1:2000 dilution; #ab6721). Blots were visualized using enhanced chemiluminescence (Thermo Fisher Scientific) and brand intensity was measured by densitometry using Quantity One software (Bio-Rad, Hercules, CA, USA).
Ab92547
Manufactured by Cell Signaling Technology
Sourced in United States
Ab92547 is a laboratory equipment product manufactured by Cell Signaling Technology. It is a tool used in scientific research. The core function of this product is to facilitate the detection and analysis of specific molecules or biological targets.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Ab166804, by Abcam (1 mentions)
Rabbit anti β actin antibody, by Cell Signaling Technology (1 mentions)
Enhanced chemiluminescence, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Protease and phosphatase inhibitor, by Merck Group (1 mentions)
β actin, by Abcam (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
E cadherin, by BD (1 mentions)
Ab45688, by Abcam (1 mentions)
Ab52903, by Abcam (1 mentions)
Dab horseradish peroxidase color development kit, by Beyotime (1 mentions)
Ab34710, by Abcam (1 mentions)
Col 1, by Abcam (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
Ab5694, by Abcam (1 mentions)
Ab86299, by Abcam (1 mentions)
6 protocols using ab92547
1
Western Blot Analysis of EMT Markers
2
Western Blot Analysis of EMT Markers
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The cells were lysed directly in an RIPA buffer (Millipore) supplemented with protease and phosphatase inhibitors (Sigma). The relative protein concentration was determined using a BCA protein assay kit (Thermo Scientific). For each lane of 8 to 10 % SDS–PAGE gel, 50 μg of cell lysate protein was loaded, separated, and transferred onto a polyvinyldifluoride (PVDF) membrane (Millipore). The membranes were then probed using specific antibodies against Matrix metallopeptidase 9 (MMP-9) (Abcam, ab38898), E-cadherin (BD Biosciences, 610,181), vimentin (Abcam, ab92547), snail protein (Cell Signaling, #3879), and β-actin (BioVision, 3598–100).
3
Histological Analysis of Kidney Fibrosis
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Kidney samples were fixed in 4% Paraformaldehyde (PFA), embedded in paraffin, and sectioned 4 μm thick for histological analysis. Masson trichrome (MTC) staining was performed to assess tissue fibrotic changes. Ten randomly selected fields (200× magnifications) from each section were analyzed by Image J (National Institutes of Health, Bethesda, MD, United States). The severity of tubulointerstitial fibrosis was indicated as the ratio of blue-stained scarred areas to the total area. For immunohistochemical studies, renal sections were incubated with antibodies against α-SMA (ab5694, Abcam, United Kingdom), Fibronectin (FN, ab45688, Abcam, United Kingdom), Collagen I (Col I, ab34710, Abcam, United States), E-cadherin (#3195, Cell Signaling Technology, United States), Vimentin (ab92547, Cell Signaling Technology, United States), Phospho-NF-κB p65 (Ser536, ab86299, Abcam, United Kingdom), Phospho-histone H3 (Ser10, #9701, Cell Signaling Technology, United States) and Phospho-smad3 (Abcam, ab52903, United Kingdom). After biotinylated secondary antibody was applied, the slides were detected by the DAB Horseradish Peroxidase Color Development Kit (Beyotime, Shanghai, China).
4
Quantifying Epithelial-Mesenchymal Transition
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After the indicated treatment, A549 or HBE cells were washed fixed with 4% methanol for 30 min, then stained with anti-Vimentin or anti-E-cadherin antibody (Abcam, ab92547, 1:1000; Cell Signaling Technology, 24E10, 1:1000) at 4 °C overnight and incubated with Cy3-conjugated or FITC-conjugated goat anti-rabbit antibody (1:200, Beyotime Institute of Biotechnology, Shanghai, China) for 1 h. The DAPI was used to stain the nucleus in cells for 10 min. All the images were acquired with the fluorescence microscope (Olympus, Tokyo, Japan).
5
Western Blot Analysis of EMT Markers
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The cell lysates were both centrifuged at 13,000 rpm (at 4°C for 5 min). After that 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) were used to segregate them and then transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). The filters were blocked with blocking buffer (Sangon Biotech) for 45 min. Membranes were incubated with primary antibodies anti-LRH1 (Abcam, ab223211), anti-E-cadherin (Abcam, ab1416), anti-N-cadherin (Abcam, ab18203), anti-Vimentin (Abcam, ab92547), anti-β-catenin (Cell Signaling Technology, D10A8)) at 4°C overnight and with secondary antibodies at 1:5,000 dilution at room temperature for 2 h, followed by incubating with β-actin (Abcam, ab179467) at room temperature for 1.5 h.
6
Protein Expression Analysis in RCC Tissues
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Cells or RCC tissues were lysed in RIPA on ice for 15 min, and the mixture was centrifuged for isolating protein samples. The concentration of protein was determined by BCA method. After adjusting protein samples to the same concentration (2ug/ul), they were denaturated, separated by SDS-PAGE, and loaded on PVDF membrane. The membrane was cut into small pieces according to the molecular size and blocked in TBST containing 5% skim milk for 2 h. They were incubated with primary (1:1000) and secondary antibodies (1:3000), followed by band exposure using ECL and grey value analyses using ImageJ software. Antibodies were purchased from Abcam (APOC1: ab198288, Wnt3a: ab219412, β-Catenin: ab32572, Vimentin: ab92547, N-cadherin: ab76011) and Cell Signaling Technology (GAPDH: 5174, MMP2: 40994, MMP9: 13667, CCND1: 55506, TCF7: 2203, anti-rabbit and anti-mouse secondary antibodies).
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