Western blot analysis in protein lysates derived by cell lines was performed as previously described 19 (link). Briefly, SDS-PAGE was conducted using 8%-12% gradient Tris-Glycine gels (Invitrogen) and loaded with 30ugs of total protein lysates. Membranes were blocked in TBS + 5% nonfat milk, primary and secondary antibodies were incubated overnight at 4° C or for 2h at RT, respectively, in TBS + 0.1% Tween-20 + 5% nonfat milk (pStat3 was incubated in TBS + 0.1% Tween-20 + 5% BSA). Primaries used were: β-actin ms (1:1000, 130065; Santa Cruz Biotech, Inc), pStat3 (Tyr705) rb (1:1000, 9145S; Cell Signaling, Inc), b-catenin rb (1/3000, ab6302; Abcam, Cambridge, MA), vimentin ms (1/2000, ab8069; Abcam), E-cadherin rb (1/500, ab53033; Abcam), ZEB1 rb (1/500, SAB3500514; Sigma-Aldrich, Saint Louis, MO), CRHR2 (1/1000, ABN433; Millipore, Temecula, CA). Horseradish peroxidase (HRP)-tagged IgG secondary antibodies were anti-mouse (sc2005; Santa Cruz Biotech, Inc) or anti-rabbit (sc2004; Santa Cruz Biotech, Inc) used at 1/2000 dilution. Chemiluminescence was detected with enhanced reagent (34080; ThermoScientific, Rockford, IL) using an Eastman Kodak Co. 440 Imaging System (Kodak, Rochester, NY). Beta-actin was used as a loading control.
Ab53033
Manufactured by Abcam
Sourced in United Kingdom
Ab53033 is a laboratory equipment product. It is a general-purpose instrument designed for scientific research applications. The core function of this product is to facilitate the performance of specific experimental procedures in a laboratory setting. No further details are available for this product while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Ab50887, by Abcam (2 mentions)
Ab6302, by Abcam (1 mentions)
Ab8069, by Abcam (1 mentions)
Anti rabbit sc 2004, by Santa Cruz Biotechnology (1 mentions)
Anti mouse sc 2005, by Santa Cruz Biotechnology (1 mentions)
P stat3 tyr705, by Cell Signaling Technology (1 mentions)
Tris glycine gel, by Thermo Fisher Scientific (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 546, by Thermo Fisher Scientific (1 mentions)
Anti gapdh, by Merck Group (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by GE Healthcare (1 mentions)
Eclipse 80i, by Nikon (1 mentions)
Ab12221, by Abcam (1 mentions)
M0879, by Agilent Technologies (1 mentions)
M7020, by Agilent Technologies (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Dojindo Laboratories (1 mentions)
Ab49117, by Abcam (1 mentions)
Ba1050, by Boster Bio (1 mentions)
Ba1054, by Boster Bio (1 mentions)
8 protocols using ab53033
1
Western Blot Analysis of Cell Lysates
2
TAFH siRNA Modulates Fibroblast and Melanoma Cells
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TAFH siRNA-treated and untreated human dermal primary fibroblasts and melanoma cells were grown on 22-mm2 glass slides to about 70% confluence. Anti-MITF (ab59232, Abcam) as primary, and Alexa Fluor 546 (Molecular Probes, Invitrogen, Eugene, OR, USA) as secondary antibodies were used for immunofluorescence analysis. Images were visualised using Nikon Eclipse 80i fluorescence microscope (Nikon Instruments Inc., USA).
For Western blot analysis, treated and untreated cells were collected by trypsin-EDTA (PAA Laboratories, Austria). Cell fractionation was carried out using NE-PER Nuclear and Cytoplasm Extraction Reagents (Thermo Scientific, Pierce, Rockford, IL, USA). Total protein concentration of nuclear and whole cell extracts was measured using BCA Protein Assay kit (Thermo Scientific, Pierce, Rockford, IL, USA). The following primary antibodies were used: anti-TAF4 (BD Biosciences, 612054), anti-CDH1 (Abcam, ab53033), anti-CDH2 (Abcam, ab12221), anti-phospho-TP53Ser15 (Cell Signaling, 9284), and anti-GAPDH (Sigma, G8795). Secondary HRP-conjugated antibodies were purchased from Abcam. Proteins were visualised using SuperSignal West Dura Chemiluminescent Substrate (Thermo Scientific, Pierce, Rockford, IL, USA).
For Western blot analysis, treated and untreated cells were collected by trypsin-EDTA (PAA Laboratories, Austria). Cell fractionation was carried out using NE-PER Nuclear and Cytoplasm Extraction Reagents (Thermo Scientific, Pierce, Rockford, IL, USA). Total protein concentration of nuclear and whole cell extracts was measured using BCA Protein Assay kit (Thermo Scientific, Pierce, Rockford, IL, USA). The following primary antibodies were used: anti-TAF4 (BD Biosciences, 612054), anti-CDH1 (Abcam, ab53033), anti-CDH2 (Abcam, ab12221), anti-phospho-TP53Ser15 (Cell Signaling, 9284), and anti-GAPDH (Sigma, G8795). Secondary HRP-conjugated antibodies were purchased from Abcam. Proteins were visualised using SuperSignal West Dura Chemiluminescent Substrate (Thermo Scientific, Pierce, Rockford, IL, USA).
3
Histological Analysis of Transgenic Fish Pancreas and Cerebellum
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Pancreas and cerebellum of eGFP-KRASG12D-positive fish and Tg(ptf1a:eGFP) controls were dissected and fixed in PBS containing 4% paraformaldehyde overnight at +4°C. Collected tissues were paraffin-embedded, cut as 1 μm slices and examined histologically using the standard H&E and Alcian Blue methods to analyze cell morphology and tissue structure. Tissue slices were immunostained with DAPI, to label cell nuclei, and antibodies anti-E-Cadherin (ab53033, Abcam, Cambridge, UK), anti-N-Cadherin (ab12221, Abcam, Cambridge, UK), anti-Vimentin (M7020, Dako, Glostrup, Denmark), and anti-PCNA (M0879, Dako, Glostrup, Denmark), according to standard procedures. A TUNEL assay protocol (Invitrogen, Carlsbad, CA) was used to detect apoptosis.
4
Saponarin Effects on Aquaporin-5 and E-Cadherin Colocalization
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Mice SMG tissue was cut into 200-μm thick slices with a tissue slicer (DSK, linear Slicer, PRO7; Dosaka EM Co., Ltd., Kyoto, Japan). The sliced tissues were kept in a 6-well culture dish with DMEM containing 10% FBS and 1% penicillin/streptomycin and stimulated with 50 μM of saponarin, schaftoside, and isoschaftoside and vehicle control for 10 min. After stimulation, tissues were fixed with 4% paraformaldehyde, and paraffin-embedded tissue sections were prepared. Immunohistochemistry was performed with mouse anti-E-cadherin (ab53033, 1:200 dilution; Abcam, Cambridge, UK) and rabbit anti-AQP5 antibody (ab15858, 1:200 dilution; Millipore Co., Ltd., Darmstadt, Germany) as primary antibodies and TRITC conjugated mouse anti-rabbit IgG (1:200 dilution; Santa Cruz Biotechnologies, CA) and AlexaFluor® 488 conjugated chicken anti-mouse IgG (1:200 dilution; Gibco Life Technologies) as secondary antibodies. DAPI (1:1,000 dilution; Dojindo Laboratories) was used for nuclear staining. The degree of colocalization between AQP5 and the plasma membrane marker E-cadherin was calculated as Pearson’s correlation coefficient with ImageJ Coloc2 plugin.(20 (link))
5
Western Blot Analysis of RUNX3, E-cadherin, and SOX2
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Cells were disrupted in lysis buffer (AR0105-30; Boster, Wuhan, China) supplemented with 0.5 mM phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 5 μg/ml pepstatin and 2.1 μg/ml aprotinin. Protein lysates were separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The membranes were incubated with rabbit antibody to RUNX3 (1:1000; ab49117; Abcam, Cambridge, UK), rabbit antibody to E-cadherin (1:1000; ab53033; Abcam), SOX2 (1:1000; 20118-1-AP; Proteintech, Chicago, IL, USA) or mouse antibody to β-Actin (1:2000; 60008; Proteintech), and subsequently incubated with HRP-conjugated goat anti-rabbit IgG (BA1054; Boster) or HRP-conjugated goat antimouse IgG (BA1050; Boster). Signals were detected using an enhanced chemiluminescence kit (Pierce, Rockford, IL, USA).
6
Western Blot Analysis of EMT Regulators
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Western blot analysis was performed by standard methods as described previously [56 (link)] using the following antibodies: rabbit anti human ZEB-1 antibody was used at a dilution of 1:500 (Abcam, ab64098); a mouse anti-human TWIST-1antibody (Abcam- ab50887) was used at a dilution of 1:250; a rabbit anti-human E-cadherin antibody (Abcam-ab53033) was used at a dilution of 1:500, a rabbit anti-human MAP4K4 antibody (Abcam-ab15583) was used at a dilution of 1:1000, a mouse anti-human JNK antibody (Santa cruze-sc7345) was used at a dilution of 1:500 and a mouse anti-human p-JNK antibody (Santa cruze-sc6254) was used at a dilution of 1:500 for overnight incubation.
7
Western Blot Analysis of E-cadherin and Twist
8
Corneal Stromal Cell Adhesion on FSCMs
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11 non-curved FSCMs, that were cut in halves, for CK3/12 (1:100; ab68260; Abcam, Cambridge, UK), Ecadherin (1:400; ab53033), integrin α6 (1:1000; ab20142), laminin-332 (1:25; ab11575; Abcam; Cambridge, UK), and additionally with ∆N P63 (1:300; sc-8609; Santa Cruz Biotechnology; CA, USA), a determinant for stemness. Corneal stromal cells were also cultured on the smooth side of FSCMs (P12261201), coated with and without rat tail collagen type I. The cells were stained for whole mount immunofluorescence with antibodies against the transmembrane receptors integrin-α6 (1:1000; ab20142) and integrin-β1 (1:50; ab52971; Abcam, Cambridge, UK), involved in cell-matrix adhesion, and counterstained for F-actin (Alexa Fluor 488 Phalloidin; A12379) to depict the cytoskeleton, and with DAPI for cell nuclei.
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