IHC staining was performed as described previously [37 (link)] using antibodies against E-cadherin (BD Biosciences, San Jose, CA, USA), β-catenin (Santa cruz, Dallas, TX, USA), Twist (Sigma-Aldrich, St. Louis, MO, USA), and Slug (Abcam, Burlingame, CA, USA). The immunoreaction was observed under a bright-field microscope at a 60× objective lens.
Twist
Manufactured by Merck Group
Sourced in United States
Twist is a versatile lab equipment designed for efficient DNA/RNA manipulation. It facilitates precise and controlled twisting, winding, and unwinding of nucleic acid strands. The core function of Twist is to enable researchers to perform essential molecular biology techniques with accuracy and reproducibility.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Merck Group (4 mentions)
E cadherin, by BD (2 mentions)
Vimentin, by BD (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Vimentin, by Abcam (2 mentions)
Gapdh, by Merck Group (2 mentions)
Vimentin, by Merck Group (2 mentions)
E cadherin, by Merck Group (2 mentions)
N cadherin, by Merck Group (2 mentions)
β catenin, by Santa Cruz Biotechnology (1 mentions)
Cyclin a2, by Santa Cruz Biotechnology (1 mentions)
Ecl reagent, by GE Healthcare (1 mentions)
Prmt1, by Merck Group (1 mentions)
Snail, by Abcam (1 mentions)
α sma, by Merck Group (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
β catenin, by BD (1 mentions)
Fibronectin, by BD (1 mentions)
Enhanced chemiluminescence system, by Bio-Rad (1 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (1 mentions)
10 protocols using twist
1
Immunohistochemical Analysis of EMT Markers
2
Western Blot Analysis of EMT Markers
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Twenty μg of whole-cell lysate was loaded onto a 7.5–15% SDS-PAGE gel. Protein lysates were transferred to PVDF membrane (Millipore) and detected by ECL reagent (GE Healthcare, Buckinghamshire, UK). The following primary antibodies were used and diluted in 2% BSA/TBST: E-cadherin (BD Bioscience, #610181, 1:5000), fibronectin (BD Bioscience, #610077, 1:2000), vimentin (BD Bioscience, #550513, 1:8000), β-catenin (BD Biosciences, #610154, 1:2000), α-SMA (Sigma, #A5228, 1:3000), β-actin (Sigma, #A1978, 1:20000), Twist (Sigma, #sc-134136, 1:1000), MMP2 (GeneTex, #GTX104577, 1:1000), MMP9 (GeneTex, #GTX100458, 1:1000), PRMT1 (Millipore, #07-404, 1:5000), ZEB1 (Santa Cruz, #sc-25388, 1:500), p21 (Santa Cruz, #sc-756, 1:1000), cyclin A2 (Santa Cruz, #sc-751, 1:2000), Snail (Abcam, #ab63371, 1:2000), Slug (Abcam, #ab27568, 1:2000), and H4R3me2as (Active Motif, #39705, 1:1000).
3
Protein Extraction and Western Blot Analysis
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RIPA lysis buffer (Thermo Fisher) with protease inhibitor (Sigma) was used to extract the total protein. The same amounts of total proteins were separated via SDS-PAGE gel and then transferred to polyvinylidene fluoride (PVDF, Millipore, Bedford, MA, USA) membranes. After blocking with 5% skim milk, the membranes were incubated with the primary antibodies[TRIM47 (Sigma); Twist, E-cadherin, N-cadherin, ZEB1, SNAI1, SLUG, and vimentin (Abcam); p53, P21, CDK4, CDK6, cyclin D1, p65, P-p65, and GAPDH (CST)] overnight at 4°C and then with the horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, CA, USA). Signals were detected with enhanced chemiluminescence system (Bio-Rad, Richmond, CA, USA).
4
Western Blot Analysis of Signaling Proteins
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Western blot analysis was done as previously described (36 (link)) with antibodies for phosphorylated FGFR (pFGFR), FGFR1, p EGFR(Y1086), EGFR, pHER2(Y1221/1222), HER2, pHER3, HER3, pAkt, Akt, Erk, cleaved PARP, PARP, Vimentin, E-cadherin, Snail, Slug, ZEB1 (Cell Signaling Technology, Danvers, MA), Twist (Sigma,St. Louis, MO), and pERK (Santa Cruz Biotechnology, CA) or β-actin (Sigma,St. Louis, MO).
5
Hypoxia-Induced Protein Expression
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NIH3T3 and primary breast skin cells were treated with 1 μM to 1 mM concentrations of CoCl2 to mimic hypoxic conditions. Cell lysates were subjected to immunoblot analyses with antibodies against HIF-1α (Genetex), total ERK (Abcam), phosphorylated ERK (Abcam), vimentin (BD Biosciences), fibronectin (Genetex), N-cadherin (Genetex), occludin (Abcam), MMP-9 (Millipore), snail (Cell Signaling), twist (Sigma-Aldrich), TIMP-1 (Epitomics) and TIMP-2 (Millipore) in combination with a horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch) and quantitative control anti-β-actin antibodies (Millipore). For immunofluorescence staining, NIH3T3 cells were subjected to hypoxic induction and stained with antibodies against HIF-1α (Genetex) and vimentin (BD Biosciences), followed by Alexa Fluor 488- and Alexa Fluor 594-conjugated secondary antibodies (Invitrogen), respectively. The signal intensity was further quantitated using ImageJ software (National Institutes of Health). Quantitation of intensity of the bands corresponding to the indicated proteins compared with β-actin in cell extracts.
6
Real-time PCR Gene Expression Analysis
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mRNA expression analyses were carried out by real-time PCR using a DNA Opticon system (MJ Research, Waltham, MA) and using DyNAmo SYBR green qPCR mix (New England Biolabs, Ipswich, MA). Standard curves were generated by serially diluting the sample expected to have the most amount of the PCR product. The yield of product for each unknown sample was calculated by applying its threshold cycle, or C(T), value to the standard curve using the Opticon Monitor analysis software (version 1.01, MJ Research, Waltham, MA). Values were normalized to corresponding 18S rRNA values and expressed as the fold increase relative to controls. Primers for HER2, HIF-1α, BCRP, GAPDH, BMI-1, Nanog and TWIST were obtained from (Sigma, St. Louis, MO or Qiagen Valencia, CA).
7
Gastric Cancer Cell Lines and Molecular Markers
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The China Facility for Type Culture Collection (CCTCC) provided the human GC cell lines MKN28, SGC7901, BGC823, and GES-1, cultivated and passaged in our hospital’s scientific research center. MTT, RPMI 1640, trypsin, and TRIzol were purchased from Invitrogen (USA); matrix metalloproteinase-2 (MMP-2), MMP-9, NM23, N-cadherin, E-cadherin, Twist, Zeb1, vimentin, large tumor suppressor kinase 2 (LATS2), and β-actin antibodies were purchased from Sigma (USA) (Table 1 ). PCR primers were designed and synthesized by Sangon Biotech Co., Ltd. (China).![]()
Antibodies’ information.
| Gene | Product number |
|---|---|
| MMP-2 | SAB5701187 |
| MMP-9 | SAB5700152 |
| NM23 | SAB4502009 |
| N-cadherin | SAB5701190 |
| E-cadherin | SAB5700789 |
| Twist | SAB5701071 |
| Zeb1 | SAB5701068 |
| vimentin | SAB5701293 |
| LATS2 | SAB5701812 |
| β-actin antibodies | A1978 |
8
Quantitative Western Blot Analysis
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After incubation with indicated factors cells were harvested in lysis buffer (0.0625 M Tris/HCl pH: 6.8, 2% SDS, 10% glycerol, 5% β-mercaptoethanol) and total protein concentration was evaluated according to Pedersen protocol. Lysates containing equal amounts of protein were separated in 10% SDS-PAGE gel and transferred onto PVDF membrane. For protein detection specific primary antibodies against: AR, p-AR(Ser210/213), E-cad, p-Ecad(Ser838/Ser840), AKT, p-AKT(Ser 473), PTEN, p-PTEN(Ser380), RAS, RAF, ERK1/2, BAX, BAD, BCL-2, SNAIL, TWIST, ZEB1 and β-actin (Sigma-Aldrich, Poland) as for referral protein were used. Secondary rabbit or mouse antibodies conjugated with HRP (1:10,000, Cell Signaling Technology, Inc.) were utilised for detection. All steps were performed as previously described [22] . In order to obtain quantitative results, the bands (representing each data point) were densitometrically scanned using SynGene Gene Tools version 4.03.0 (Synoptics Ltd Beacon House, Nuffield Road Cambridge, CB4 1TF, UK). β-actin was used as a control. Protein level within the control group was arbitrarily set as 1. The procedure was performed as previously described [24] (link).
9
Protein Expression Analysis by Western Blot
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Cells were lysed on ice in lysis buffer [10 mM Tris-HCl (pH 7.4), 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM Na4P2O7, 2 mM Na3VO4, 1% Triton X-100, 10% glycerol, 0.1% SDS, 0.5% deoxycholate] (Invitrogen, Camarillo, CA, USA) supplemented with 1 mM phenylmethylsulfonyl fluoride and protease inhibitor cocktail (Sigma, St. Louis, MO, USA). Whole cell extracts (20 µg/lane) were electrophoresed using 10% SDS-polyacrylamide gels and electrotransferred to polyvinylidene fluoride membranes (Millipore, Schwalbach, Germany) as described previously.3 (link) The following antibodies were used for Western blotting: anti-HIF-1α (1:1000), -E-cadherin (1:500), -N-cadherin (1:500), -vimentin (1:1000), -Twist (1:1000), -Slug (1:1000), and GAPDH (1:1000). All antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).
10
Quantitative Protein Expression Analysis
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The proteins were separated according to their molecular weight via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a PVDF membrane (GE Healthcare, Amersham, UK), and probed with mouse monoclonal antibodies or rabbit polyclonal antibodies specific for the proteins of interest. The blots were developed using the enhanced chemiluminescence (ECL) technique (pERKinElmer, Boston, MA, USA) according to the manufacturer's instructions, and the level of each protein was quantified and compared (DKK-1, VE-cadherin and Vimentin: Abcam, Cambridge, UK; VEGFR2, β-actin, AKT, pAkt, ERK, pERK, GSK3b and N-cadherin: Cell Signaling, Danvers, MA, USA; Twist: Millipore, Billerica CA, USA). To detect secreted DKK-1, an enzyme-linked immunosorbent assay (ELISA; R&D Systems, Minneapolis, MN, USA) was performed according to the manufacturer's instructions.
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