Total protein was extracted from cells, lung tissues of patients, and xenograft tumour tissues of mice using RIPA lysis buffer (Elabscience). Total protein was subjected to 10% SDS-PAGE, and then transferred to polyvinylidene difluoride membranes. Polyvinylidene difluoride membranes were blocked with 5% non-fat milk in TBST for 1 h at 37 °C. After blocking, the membrane was incubated with primary antibodies (CHCHD4, cat no. 21090-1-AP, Proteintech, Wuhan, China; Bcl-2, cat no. 26593-1-AP, Proteintech; Cleaved-PARP, cat no. #5625, Cell Signalling, USA; Bax, cat no. 50599-2-Ig, Proteintech; E-cadherin, cat no. A20798, Abclonal, Wuhan, China; N-cadherin, cat. ab280375, Abcam, USA; vimentin, cat no. 10366-1-AP, Proteintech; MYC, cat no. 10828-1-AP, Proteintech, USA; Snai1, cat no. 13099-1-AP, Proteintech; Snai2, cat no. 12129-1-AP, Proteintech; and Twist1, cat no. A7314, Abclonal,; GAPDH, cat no. 10494-1-AP, Proteintech) overnight at 4 °C. Membranes were then incubated with horseradish peroxidase-conjugated goat polyclonal anti-rabbit IgG secondary antibodies (cat. no. ab150077; Abcam) for 1 h at room temperature. The target bands were visualised using a chemiluminescence kit (Beyotime).
Snai1
Manufactured by Proteintech
Sourced in United States
SNAI1 is a protein that functions as a transcriptional repressor. It plays a role in the epithelial-mesenchymal transition process.
Automatically generated - may contain errors
Lab products found in correlation
Vimentin, by Proteintech (6 mentions)
Gapdh, by Proteintech (5 mentions)
E cadherin, by Proteintech (5 mentions)
N cadherin, by Proteintech (3 mentions)
E cadherin, by Cell Signaling Technology (3 mentions)
Vimentin, by Cell Signaling Technology (3 mentions)
Bcl2 associated x (bax), by Proteintech (2 mentions)
X box binding protein 1 xbp1, by Proteintech (2 mentions)
Cavelino 1, by Proteintech (2 mentions)
β actin, by Proteintech (2 mentions)
Glyat, by Abcam (2 mentions)
Pi3k p58, by Proteintech (2 mentions)
N cadherin, by Cell Signaling Technology (2 mentions)
P akt ser473, by Signalway Antibody (2 mentions)
Anti β actin, by Proteintech (2 mentions)
Ecl detection kit, by Thermo Fisher Scientific (2 mentions)
N cadherin, by ABclonal (1 mentions)
Chchd4, by Proteintech (1 mentions)
Snai2, by Proteintech (1 mentions)
Ripa lysis buffer, by Elabscience (1 mentions)
14 protocols using snai1
1
Protein Extraction and Western Blot Analysis
2
Antibodies for Cellular Signaling Analysis
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Antibodies against KIFC3 were purchased from Abcam (Cambridge, MA, United States). Antibodies against proliferating cell nuclear antigen (PCNA), matrix metalloproteinase (MMP)-2, MMP-9, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), p21, Cyclin A2, Cyclin E1, CDK2, E-cadherin, N-cadherin, Vimentin, Snai1 were purchased from Proteintech (Rosemont, IL, United States). Antibodies against p53, TWIST1 were obtained from Affinity (Cincinnati, United States). Antibodies against total phosphatidylinositol 3-kinase (t-PI3K), phosphorylated phosphatidylinositol 3-kinase (p-PI3K), total AKT (t-AKT), phosphorylated Akt (p-AKT), total mammalian target of rapamycin (t-mTOR), phosphorylated mammalian target of rapamycin (p-mTOR) were obtained from Cell Signaling Technology (Danvers, MA, United States). Antibodies against PCNA, GAPDH, E-cadherin, N-cadherin, Vimentin, were used at a working concentration of 1:5000, and the other antibodies were used at a working concentration of 1:1000 and were stored at 4°C. The secondary antibodies were purchased from Proteintech (Rosemont, IL, United States) and used at a dilution ratio of 1:10,000.
3
Western Blot Analysis of EMT Markers
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Cells were lysed in RIPA buffer containing 1 mM phenylmethylsulfonyl fluoride (PMSF) (both Beyotime Institute of Biotechnology). The prepared protein samples were quantified using a bicinchoninic protein assay kit (Beijing Solarbio Science and Technology Co., Ltd.). Equal amounts of protein (40 µg/lane) were separated by 12% SDS-PAGE and then transferred to a PVDF membrane. The membranes were firstly blocked with 5% bovine serum albumin (Beijing Solarbio Science and Technology Co., Ltd.) at room temperature for 1 h and then incubated with primary antibodies against XB130 (1:1,000; cat. no. ab106433; Abcam), N-cadherin (1:5,000; cat. no. 22018-1-AP; ProteinTech Group, Inc.), SNAI1 (1:2,000; cat. no. 26183-1-AP; ProteinTech Group, Inc.), β-catenin (1:5,000; cat. no. 51067-2-AP; ProteinTech Group, Inc.) and GAPDH (1:5,000; cat. no. 10494-1-AP; ProteinTech Group, Inc.) overnight at 4°C, and finally with horseradish peroxidase conjugated secondary antibodies (1:5,000; cat. no. SA00001-2; ProteinTech Group, Inc.) at room temperature for 1 h. The specific protein bands were visualized using an ECL reagent (EMD Millipore).
4
Detailed Characterization of SC66 and CHIR-99021 Effects
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SC66 (purity of 99.88%) was purchased from Selleck (Cas: 871361‐88‐5, China) and dissolved in dimethyl sulfoxide (DMSO) to get to the required concentration. CHIR‐99021, a selective GSK‐3 inhibitor (purity of 99.76%), was purchased from MedChemExpress (Cas: 252917‐06‐9, China). The following antibodies were used in the present study: P‐AKT (66444‐1‐Ig, Proteintech), AKT (60203‐2‐Ig, Proteintech), P‐GSK‐3β (#9323, CST), GSK‐3β (#9832, CST), P‐β‐catenin (#9567, CST), β‐catenin (#8480, CST), SNAI1 (13099‐1‐AP, Proteintech), MMP2 (10373‐2‐AP, Proteintech), E‐cadherin (20874‐1‐AP, Proteintech), vimentin (10366‐1‐AP, Proteintech), cyclin D1 (60186‐1‐Ig, Proteintech), Bcl‐2 (#15071, CST), Bax (60267‐1‐Ig, Proteintech), cleaved‐Caspase‐3 (#9664, CST) and GAPDH (60004‐1‐Ig, Proteintech). More detailed antibody information is shown in Table 1 .
5
Immunocytochemistry Analysis of EMT Markers
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As previously explained (Yang et al., 2017 (link)), immunocytochemistry was carried out following exposure of HCT116 cells to cisatracurium. In summary, HCT116 cells were incubated in PBS containing 4% para-formaldehyde at 25°C for 20 min. For permeabilization, HCT116 cells were further maintained in PBS containing 0.5% Triton X-100 at 4°C for 10 min. After blocking using 3% BSA reagent, cells were exposed to primary antibodies for SLUG, SNAI1, E-Cadherin, and CALD1 (Proteintech). FITC conjugated secondary antibody (Invitrogen) was added and then stained with 1 μg/ml of DAPI for 60 s. Images of samples were visualized and captured using a fluorescence microscope (Olympus BX83, Japan).
6
ER Stress Pathway Activation in PC Cells
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Whole protein lysates were prepared from transfected PC cells. Samples were loaded onto 10% SDS-polyacrylamide gels, transferred to PVDF membranes and incubated with primary antibodies: CRT (Abcam), IRE1α (CST), PREK (CST), phosphorylation PKR-like endoplasmic reticulum kinase (p-PERK, CST), ATF-6 (CST), ZO-1 (Proteintech), ZEB1 (Proteintech), N-cadherin (Proteintech), E-cadherin (Proteintech), Vimentin (Proteintech), phosphorylation extracellular regulated protein kinases (pERK, CST), extracellular regulated protein kinases (ERK, CST), X-box-binding protein 1 (XBP1, Proteintech), Snai1 (Proteintech), Slug (CST), Cavelino-1 (Proteintech), GAPDH (Proteintech) and β-actin (Proteintech) antibodies overnight at 4 °C. Then, membranes were incubated with secondary antibodies (Santa Cruz, CA, UK) and finally detected with an ECL detection kit (Thermo Scientific, Rockford, IL, USA). The experiments were repeated for 3 times.
7
Antioxidant Regulation of TGF-β Signaling
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HA, N-acetylcysteine (NAC), diphenylene iodonium (DPI), and dimethyl sulfoxide (DMSO) were provided by Sigma Aldrich company (St. Louis, MO, USA) and MG132 was obtained in Aladdin (Shanghai, China). Sulforaphane (SFN) was from TRC company (North York, ON, Canada). Antibodies against phosphorylated SMAD2 (p-SMAD2), p-SMAD3, SMAD2, SMAD3, SMAD4, CUL3, tissue inhibitor of metalloproteinases-1 (TIMP1), and ubiquitin (Ub) were obtained by CST company (Danvers, MA, USA). Antibodies containing collagen-I (COL1A1), E-cadherin (CDH1), alpha-smooth muscle actin (ACTA2), vimentin (VIM), heme oxygenase-1 (HO1), KEAP1, SNAI1, matrix metalloproteinase-9 (MMP9), NAD(P)H quinone dehydrogenase (NQO1), β-ACTIN, histone H3, and secondary antibodies were purchased from Proteintech (Chicago, IL, USA). Antibodies against NADPH oxidase 4 (NOX4) and NRF2 were provided by Abcam company (Cambridge, UK).
8
Protein Expression Analysis of EMT Markers
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Cells were lysed in ice-cold RIPA buffer containing protease inhibitors. Protein samples (40 µg) were loaded for electrophoresis on 8–12% denaturing SDS-PAGE gels. The blots were probed with the corresponding primary antibodies overnight at 4 °C, followed by incubation with the appropriate secondary antibodies at room temperature for 1 h. The following primary antibodies were used for detection: ATF3 (1:2000, #ab207434, Abcam. Inc), Twist1(1:2000, #25465-1-AP, Proteintech. Inc), Slug (1:2000, #12129-1-AP, Proteintech. Inc), Snai1(1:2000, #13099-1-AP, Proteintech. Inc), and GAPDH (1:2000, # YM3029, Immunoway. Inc).
9
Western Blot Analysis of EMT Markers
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Cell lysates were produced using a RIPA lysis solution containing a protease inhibitor. The proteins’ concentration was established using a BCA kit from Beijing, China’s Beyotime Biotechnology, before they were separated on polyacrylamide gels (PAGE) with 10% SDS and transferred onto polyvinylidene difluoride (PVDF) membranes. PIK3CD (1:1000, Zenbio, R382122), E-cadherin (1:1000, Affinity, AF0131), vimentin (1:1000, proteintech, 10,366-1-AP), SNAI1(1:1000, proteintech, 13,099-1-AP) and GAPDH (1:1000, Zenbio, 380,626) were then incubated with the hydrogels at 4 ℃. The membranes were incubated for 45 min with a second antibody (1:4000, proteintech, SA00001-2) in combination with horseradish peroxidase following three TBST washes. We imagined and measured the protein bands.
10
Immunohistochemical Analysis of EMT Markers
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The tissues were sectioned, treated with 3% H2O2, and then incubated in 5% goat antiserum. Non-serial tissue sections were incubated with the primary antibodies against E-Cadherin (1:200; Abcam, USA), ZEB1 (1:100; Abcam, USA), TWIST (1:200; Boster, China), SNAI1 (1:100; Proteintech, USA), Vimentin (1:500; Abcam, USA) overnight, and then with biotin-labeled secondary antibodies. Streptavidin-peroxidase complex was added, and the sections were stained with 3,3′-diaminobenzidine (Maixin Biotech, Fuzhou, China) prior to microscopy analyses. All sections were independently scored by three experienced pathologists. Scoring was based on the percentages of positive cells with different staining intensities.
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