For the westernblot assay, cells were harvested in ice-cold PBS 48 h after transfection and lysed on ice in cold-modified radioimmunoprecipitation buffer supplemented with protease inhibitors. Protein concentration was determined using the BCA Protein Assay Kit (Bio-Rad, CA, USA) and equal amounts of protein were analyzed by SDS-PAGE. Gels were electroblotted onto nitrocellulose membranes (Millipore, WI, USA). After blocked with 5% non-fat dry milk in Tris-buffered saline containing 0.1% Tween-20 2 h, membranes were incubated at 4°C over night with primary antibody. Primary antibodies used were anti-STAT3, anti-p-STAT3, anti-MMP2, anti-BCL2, anti-E-cadherin, anti-KIRT1, anti-SNAI2, anti-N-cadherin (Cell Signaling, USA) and GAPDH (Zhong-Shan JinQiao, China). Then, membranes were incubated with respective second antibodies and detected by peroxidase-conjugated secondary antibodies using the enhanced chemiluminescence system (ECL) (Millipore, WI, USA). The experiment was repeated three times.
Anti snai2
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-SNAI2 is a primary antibody that recognizes the SNAI2 protein. SNAI2 is a transcription factor involved in the regulation of epithelial-mesenchymal transition (EMT) and cell migration. The antibody can be used to detect and study the expression of SNAI2 in various experimental systems.
Automatically generated - may contain errors
Lab products found in correlation
Anti n cadherin, by Cell Signaling Technology (2 mentions)
Anti β catenin, by Cell Signaling Technology (2 mentions)
Anti vimentin, by Cell Signaling Technology (2 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Anti mmp2, by Cell Signaling Technology (1 mentions)
Anti bcl 2, by Cell Signaling Technology (1 mentions)
Anti e cadherin, by Cell Signaling Technology (1 mentions)
Anti p stat3, by Cell Signaling Technology (1 mentions)
Bca protein assay kit, by Bio-Rad (1 mentions)
Anti c myc, by Cell Signaling Technology (1 mentions)
Anti sox2, by Cell Signaling Technology (1 mentions)
Anti oct4, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Anti nanog, by Cell Signaling Technology (1 mentions)
Anti klf4, by Santa Cruz Biotechnology (1 mentions)
Anti epcam, by Santa Cruz Biotechnology (1 mentions)
Anti aldh1, by Santa Cruz Biotechnology (1 mentions)
Anti aldh2, by Santa Cruz Biotechnology (1 mentions)
Anti fosl2, by Cell Signaling Technology (1 mentions)
Conjugate goat anti rabbit secondary antibody, by Cell Signaling Technology (1 mentions)
9 protocols using anti snai2
1
Western Blot Analysis of Protein Markers
2
Western Blotting Analysis of Stem Cell Markers
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The experimental procedure for Western blotting analysis that was used in this study was followed as described in our previous study [29 (link)]. The horseradish peroxidase-conjugated IgG that was anti-mouse and anti-rabbit (Thermo Fisher Scientific, New York, NY, USA) was used in this study. GAPDH was used as the control and for quantification. Antibodies purchased from Santa Cruz, USA: anti-EPCAM (1:1000, sc-25308), anti-ALDH1 (1:300, sc-374149), anti-ALDH2 (1:300, sc-100496), anti-KLF4 (1:1000, sc-166100), anti-GAPDH (1:1000, sc-47724). Antibodies purchased from Cell Signaling Technology, USA: anti-SNAI2 (#9585, 1:1000), anti-SOX2 (#3579, 1:1000), anti-OCT4 (#2840, 1:1000), anti-NANOG (#4903, 1:1000), anti-β-catenin (#8480, 1:1000), anti-c-Myc (#18583, 1:1500 dilution). Anti-PCNA (#60097-1-Ig, 1:5000) were purchased from Proteintech. The signal intensity was quantified using the protein imprinting imaging system (Tanon 5200, Better Tanon, Shanghai, China).
3
Western Blot Analysis of Epithelial-Mesenchymal Transition
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The cells were harvested and lysed by RIPA buffer for 30 min at 4°C. 50 µg heat-denatured proteins were loaded into 15% SDS–PAGE for SDS-polyacrylamide gel electrophoresis, and then transferred to polyvinylidene difluoride membrane for Western blotting analysis. After blocking nonspecific binding sites with 5% (wt/vol) nonfat milk, 0.1% (vol/vol) Tween-20 diluted in Tris (pH 7.8)-buffered saline, rabbit polyclonal anti-FOSL2 (Cell Signaling, 1:1000 dilutions), anti-ERK1/2 (p44/42 MAPK, Cell Signaling, 1:1000 dilutions), anti-phospho-ERK1/2 (phospho-p44/42 MAPK, T202/T204, Cell Signaling, 1:1000 dilutions), anti-SNAI2 (Cell Signaling, 1:1000 dilutions) and anti-GAPDH (Cell Signaling, 1:1000 dilutions) primary antibody was used to incubate overnight at 4°C. Then, HRP (horseradish peroxidase) conjugate goat-anti-rabbit secondary antibody (Cell Signaling, 1:1000 dilutions) was used to incubate for 4 hrs. The bound antibodies were detected using ECL Plus Western Blotting Detection system (GE Healthcare). GAPDH was used as an internal control.
4
Detecting SNAI2 and USP20 Proteins
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Primary antibodies for Western blotting and immunohistochemical staining included anti-SNAI2 (Cell Signaling Technology 9585), anti-USP20 (Proteintech 17491-1-AP), anti-β-actin (Abcam ab6276, clone AC-15), anti-HA (Roche 11867423001), anti-ubiquitin (Santa Cruz Biotechnology sc-271289), anti-SNAI1 (Cell Signaling Technology 3895S), anti-MYC (9E10) (Santa Cruz Biotechnology), and anti-β-catenin (Cell Signaling Technology 9585).
The negative control siRNA (D-001810-01-05), SNAI2 siRNA (J-017386-06-0002), and the human DUB siRNA library GU-104705) were purchased from GE Healthcare Dharmacon. Individual USP20 siRNAs were synthesized or purchased from Sigma (siRNA#1 sequence: GGACAAUGAUGCUCACCUA, and siRNA#2 siRNA ID: SASI_Hs02_00324906).
The negative control siRNA (D-001810-01-05), SNAI2 siRNA (J-017386-06-0002), and the human DUB siRNA library GU-104705) were purchased from GE Healthcare Dharmacon. Individual USP20 siRNAs were synthesized or purchased from Sigma (siRNA#1 sequence: GGACAAUGAUGCUCACCUA, and siRNA#2 siRNA ID: SASI_Hs02_00324906).
5
STC2 Overexpression and Knockdown
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The human STC2 expression plasmid (Cat#RC200537, OriGene) and shRNA plasmid kit (Locus ID 8614, Cat#TR309053, OriGene) were transfected using Xfect (Takara Bio Company according to the manufacturer’s protocol. Briefly, Xfect polymer was added into each plasmid (5 µg) in Xfect reaction buffer and incubated for 10 min at room temperature to allow nanoparticle complexes to form. Then, the entire complex solution was added dropwise to cells in 6 well plates. After transfection, cells were selected by independently exposing them to G418 or puromycin for 3–4 weeks. The following primary antibodies were used: rabbit polyclonal anti-STC2 (ab262857, Abcam), rabbit monoclonal anti-actin (Cat#4970, Cell Signaling Technology), anti-SNAI2 (Cat#9585, Cell Signaling Technology), anti-Vimentin (Cat#5741, Cell Signaling Technology). Human recombinant STC2 (Cat#9405-SO) was purchased from R&D Systems. LGK974 (S7143), LY3214996 (S8534), Rapamycin (S1039), LY294002 (S1105), and SB202190 (S1077) were purchased from Selleckchem.
6
Multicolor Flow Cytometry and Immunohistochemistry
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For flow cytometry, antibodies against mouse antigens were purchased from Biolegend (San Diego, CA) or BD Biosciences (Franklin Lakes, NJ) unless otherwise specified and included CD24-PE (M1/69), CD31-B (MEC13.3), TER-119-B (TER-119), CD45-B (30-F11), CD29-FITC (HMB1-1), CD61-APC and streptavidin-APC-Cy7. For immunohistochemistry, the following antibodies were used: anti-SMA (1A4; Sigma), anti-p63 (4A4), K8/18 (Troma-1; Developmental Studies Hybridoma Bank, Iowa City), anti-Keratin 5 (Covance, Emeryville, CA), anti-Keratin 14 (LL002; Novocastra, UK), anti-E-cadherin, anti-ASAP1 (Rockland Immunochemicals Inc., Limerick, PA), anti-PROX1 (ab37128; Abcam, Cambridge, UK), anti-SNAI2 (#9585 Cell Signaling Technology, Massachussetts, USA). Secondary antibodies included biotin-conjugated goat anti-rabbit IgG, rabbit anti-rat IgG and goat anti-mouse IgG (Vector Laboratories, Burlingame, CA).
7
Quantitative Protein Expression Analysis
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Total protein was isolated by freshly prepared RIPA Lysis Buffer and homogenized with 26 G syringe. Protein concentrations were determined with Bradford assay. 60 μg of protein sample were run on SDS-Gel and transferred to PVDF membranes for detection by chemiluminescence by using Vilber Fusion SL Imaging System. Images were quantified by ImageJ Gel Analysis Tool. Intensity values of related bands were normalized to values of Beta-actin housekeeping protein.
Student's t-test was used for statistical calculations. Following primary antibodies were used: anti-β-actin (Abcam, AB75186, 1/3000), anti-CYR61 (Abcam, AB127988, 1/400), anti-HEY2 (Abcam, AB184246, 1/ 500), anti-SNAI2 (Cell Signaling Technology, 9585S, 1/1000), anti-Vimentin (Cell Signaling Technology, 5741 P, 1/1000).
Student's t-test was used for statistical calculations. Following primary antibodies were used: anti-β-actin (Abcam, AB75186, 1/3000), anti-CYR61 (Abcam, AB127988, 1/400), anti-HEY2 (Abcam, AB184246, 1/ 500), anti-SNAI2 (Cell Signaling Technology, 9585S, 1/1000), anti-Vimentin (Cell Signaling Technology, 5741 P, 1/1000).
8
Protein extraction and analysis in cancer cells
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Total protein
was extracted using RIPA lysis buffer. Conditioned medium (CM) was
collected from 3.5 × 105 MCF10A cells, 4 × 105 MDA MB 231, and MCF7 cells seeded on a 6-well plate after
2 days of incubation with serum-free medium. Protein isolation from
CM was done by methanol/chloroform precipitation as described previously.19 (link) The amount of protein was quantified with Bradford
Assay. The total amount of protein of 60 μg from cell lysates
and 20 μg from CM were separated via SDS-gel and transferred
onto polyvinylidene fluoride (PVDF) membranes. After blocking with
5% skim milk, the membranes were incubated with the following antibodies:
anti-β-actin (Abcam, ab75186, 1:2000), anti-N-cadherin (Cell Signaling Technology, 13116T, 1:1000), anti-SNAI2
(Cell Signaling Technology, 9585S, 1:1000), anti-SEMA6D (total cell
lysates) (Abcam, ab191169, 1:250), and anti-SEMA6D (CM) (R&D,
AF2095-SP, 1:1000). Following secondary antibody incubation, the detection
was done using Vilber Fusion SL Imaging System.
was extracted using RIPA lysis buffer. Conditioned medium (CM) was
collected from 3.5 × 105 MCF10A cells, 4 × 105 MDA MB 231, and MCF7 cells seeded on a 6-well plate after
2 days of incubation with serum-free medium. Protein isolation from
CM was done by methanol/chloroform precipitation as described previously.19 (link) The amount of protein was quantified with Bradford
Assay. The total amount of protein of 60 μg from cell lysates
and 20 μg from CM were separated via SDS-gel and transferred
onto polyvinylidene fluoride (PVDF) membranes. After blocking with
5% skim milk, the membranes were incubated with the following antibodies:
anti-β-actin (Abcam, ab75186, 1:2000), anti-N-cadherin (Cell Signaling Technology, 13116T, 1:1000), anti-SNAI2
(Cell Signaling Technology, 9585S, 1:1000), anti-SEMA6D (total cell
lysates) (Abcam, ab191169, 1:250), and anti-SEMA6D (CM) (R&D,
AF2095-SP, 1:1000). Following secondary antibody incubation, the detection
was done using Vilber Fusion SL Imaging System.
9
Immunoblotting Antibody Validation
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The following primary antibodies were used: anti-ACTB (Cell Signaling Technology), anti-PDSS1 (Novus), anti-VCL (Cell Signaling Technology), anti-SNAI2 (Cell Signaling Technology), anti-STAT3 (Cell Signaling Technology), anti-phospho (p)-STAT3-705 (Cell Signaling Technology), anti-Flag (Sigma), anti-HA (Cell Signaling Technology), anti-JAK2 (Cell Signaling Technology), antip-JAK2 (Cell Signaling Technology), anti-HDAC1 (Cell Signaling Technology), anti-a-tubulin (Cell Signaling Technology), anti-p-CAMK2A (Abcam), and anti-CAMK2A (Proteintech). Horseradish peroxidase-conjugated anti-mouse (1:5,000 dilution) and anti-rabbit secondary antibodies (1:5,000 dilution) were purchased from Jackson ImmunoResearch.
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