BEAS-2B cells were washed with ice-cold PBS and incubated with 0.15 ml of modified RIPA lysis buffer with protease and phosphatase inhibitor cocktails 1 and 2 (Sigma-Aldrich, St. Louis, MO). Protein concentration was determined by the BCA protein assay kit (Sigma-Aldrich, St. Louis, MO) according to the manufacturer’s instructions. Each sample of protein containing 15 μg of protein was mixed with equal volume of Laemmli buffer containing 10% 2-mercaptoethanol. Proteins were resolved on 10-20% polyacrylamide gel (BioRad, Hercules, CA). Proteins were transferred onto a nitrocellulose membrane (BioRad, Hercules, CA), which were subsequently blocked with 5% non-fat dry milk for one hour. The membrane was then incubated overnight at 4°C with E-cadherin (ab15148, Abcam, Cambridge, MA), Snail (NBP1-19529, Novus, Littleton, CO), and Vimentin (sc-6260, Santa Cruz, Dallas, Texas). Following, a horseradish peroxidase-conjugated secondary antibody (Novus, Littleton, CO) was added to the membrane. The protein expression was detected by ECL chemiluminescence detection reagents (Bio-Rad, Hercules, CA). The immunoreactivity was captured by the ChemiDoc™ MP System (Bio-Rad Laboratories, Hercules, CA). Membranes were stripped and reprobed for N-cadherin and GAPDH. Results were normalized against housekeeping gene GAPDH.
Vimentin sc 6260
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Vimentin sc-6260 is a primary antibody product manufactured by Santa Cruz Biotechnology. It is designed to detect the vimentin protein, which is a type III intermediate filament protein found in various non-epithelial cells.
Automatically generated - may contain errors
Lab products found in correlation
E cadherin sc 8426, by Santa Cruz Biotechnology (5 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (4 mentions)
Twist ab50887, by Abcam (3 mentions)
N cadherin sc 7939, by Santa Cruz Biotechnology (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Snail ab167609, by Abcam (2 mentions)
Slug ab27568, by Abcam (2 mentions)
P mtor ab109268, by Abcam (2 mentions)
Fibulin 3 sc 33722, by Santa Cruz Biotechnology (2 mentions)
N cadherin sc 59987, by Santa Cruz Biotechnology (2 mentions)
Ripa buffer, by Merck Group (2 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Protease and phosphatase inhibitor cocktails 1 and 2, by Merck Group (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Novus Biologicals (1 mentions)
Bca protein assay kit, by Merck Group (1 mentions)
Chemidoc mp system, by Bio-Rad (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
E cadherin, by Abcam (1 mentions)
Ecl substrate, by GE Healthcare (1 mentions)
Smad2 3, by Cell Signaling Technology (1 mentions)
13 protocols using vimentin sc 6260
1
Epithelial-Mesenchymal Transition Protein Analysis
2
Immunoblotting Analysis of Apoptosis and Cell Cycle Markers
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Harvested cell pellets were lysed in (100–200 μl) of RIPA buffer (Sigma-Aldrich) and then proteins were extracted and quantified. 10 µg of extracted lysate was resolved in SDS-polyacrylamide gel electrophoresis (SDS-PAGE), followed by electroblotting onto methanol-activated PVDF membranes (Millipore, USA) using a semidry transfer unit (ATTO, Japan). Immunoblotting was performed with antibodies against caspase-9 (sc-7885), caspase-3 (sc-7148), Cyclin D1 (sc-450), Cyclin A (sc-239), Vimentin (sc-6260), β-catenin (sc-7963), CDK4 (sc-260), CDK2 (sc-163), PARP 1/2 (sc-7150), ATR (SC-28901), pATR (sc-109912), purchased from Santa Cruz. hnRNP-K (#4675), MMP-9 (#2270), p21WAF1 (#2947), SMAD-2/3 (#8685) and CHK-1 (#2345) were procured from Cell Signaling Technologies. Antibody for CARF (rabbit polyclonal) was generated endogenously in the laboratory. The immunoblots were incubated with horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit antibodies (Santa Cruz Biotechnology) and detected using ECL substrate (GE Healthcare, NJ, USA). Densitometric quantitation of the representative immunoblots was carried out using the ImageJ software from NIH (National Institute of Health). All the experiments were performed in triplicate.
3
Molecular Characterization of HFL1 Cells
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The HFL1 cell line was purchased from ATCC (Manassas, VA, USA). HAM’s12 medium, L-glutamine, Penicillin-Streptomycin solution, 1,25(OH)2D3, Montelukast sodium, Leukotriene D4, goat anti-rabbit IGG, goat anti-mouse IGG, protease inhibitor cocktail, RIPA buffer and CYSLTR1 antibody were purchased from Merck (Darmstadt, Germany). Fetal bovine serum was purchased from Genos (Lodz, Poland). Beclomethasone 17-Propionate and primary antibodies such as E-cadherin (sc-8426), MMP-9 (sc-393859), ADAM33 (sc-514055), α-Actin (sc-32251), GAPDH (sc-47724) and Vimentin (sc-6260) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Non-essential amino acids solution, TGFB1 Recombinant Human Protein, TRIzolTM Plus RNA Purification Kit, High-Capacity cDNA Reverse Transcription Kit, TaqManTM Gene Expression Master Mix, TaqMan™ gene expression assays: ACTA2 (Hs00426835_g1), MMP-9 (Hs009557562_m1), VIM (Hs00958111_m1), CysLTR1 (Hs00272624_s1), ADAM33 (Hs00905552_m1), CDH1 (Hs01023895_m1) and GAPDH (Hs02786624_g1) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Lastly, 4–20% ExpressPlus PAGE gel and MOPS Running buffer powder were purchased form GenSignal (Poznan, Poland).
4
Western Blot Analysis of EMT Markers
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Cells were lysed on ice in RIPA (radio-immunoprecipitation assay) buffer with 1 mM PMSF (phenylmethylsulfonyl fluoride). From the cell lysate, 40 μg of total protein was loaded into the wells of the SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel elec trophoresis) gel, along with a molecular weight marker. Electrophoresis was carried out for 1–2 h at 100 V, followed by transfer to PVDF (polyvinyl difluoride) membranes, which were then blocked with 5% BSA (bovine serum albumin). The membranes were then incubated overnight at 4°C with primary antibodies (E-cadherin sc-8426, N-cadherin sc-7939, vimentin sc-6260, Santa Cruz; fibulin-4 ab125073, Snail ab167609, Slug ab27568, Twist ab50887, PI3K ab86714, p-PI3K ab182651, AKT ab8805, P-AKT 38449, mTOR ab32028, p-mTOR ab109268; Abcam) at working dilutions of 1:1000. After washing the membranes thrice with TBST for 5 min each, the membranes were incubated with conjugated secondary antibody diluted to 1:1000, at room temperature for 1 h. Blots were developed using the enhanced chemiluminescence method (Pierce™ ECL Western Blotting Substrate; Thermo Fisher Scientific, Inc.).
5
Western Blot Analysis of EMT Markers
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Using radioimmunoprecipitation assay buffer supplemented with 1 mM phenylmethylsulfonyl fluoride, cells were lysed on ice. Protein samples (40 μg/lane) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to polyvinyl difluoride membranes and blocked with 5% bovine serum albumin. These membranes were then cultured with primary antibodies (E-cadherin sc-8426, N-cadherin sc-7939, vimentin sc-6260, Santa Cruz; HMGB1 ab18256, Snail ab167609, Twist ab50887, Abcam) with working dilutions 1:1,000 at 4°C overnight. The next day, these membranes were incubated with secondary antibody with working dilutions 1:2,000 at room temperature for 1 h, and blots were developed using the enhanced chemiluminescence method (Pierce™ ECL Western Blotting Substrate; Thermo Fisher Scientific, Inc.).
6
Western Blot Analysis of EMT Markers
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Cells were lysed on ice in RIPA (radio-immunoprecipitation assay) buffer containing 1 mM phenylmethylsulfonyl fluoride (PMSF). Samples (40-μg protein per lane) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinyl difluoride (PVDF) membranes, and blocked with 5% bovine serum albumin (BSA). Membranes were then incubated with primary antibodies (E-cadherin sc-8426, N-cadherin sc-7939, Vimentin sc-6260, Santa Cruz; fibulin-4 ab125073, Snail ab167609, Slug ab27568, Twist ab50887, β-catenin ab32572, C-myc ab32072, Cyclin D1 ab134175, Notch1 intracellular domain (NICD) ab83232, HES 1 ab71559, HEY L ab154077, Abcam) at working dilutions of 1:1000 at 4°C overnight. On the next day, membranes were incubated with secondary antibody for 1 hour at room temperature, and blots were developed using the enhanced chemiluminescence method (Pierce™ ECL Western Blotting Substrate; Thermo Fisher Scientific, Inc.).
7
Western Blot Analysis of EMT Markers
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Target cells were lysed on ice using radioimmunoprecipitation assay (RIPA) lysis buffer with phenylmethylsulfonyl fluoride (PMSF) as a serine protease inhibitor (RIPA:PMSF = 100:1). After determining the protein concentration by the Bicinchoninic Acid (BCA) method, protein samples (40 µg) were separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride (PVDF) membranes, and blocked for 1 h with Tris-buffered saline containing Tween 20 (TBST) and 5% bovine serum albumin (BSA). The membranes were then incubated on a shaking bed with primary antibodies (fibulin-3 sc-33722, E-cadherin sc-8426, N-cadherin sc-59987, Vimentin sc-6260, Santa Cruz; Snail ab167609, Slug ab106077, Twist ab50887, Zeb 2 ab138222, PI3K ab86714, p-PI3K ab182651, AKT ab8805, P-AKT ab38449, mTOR ab32028, p-mTOR ab109268, Abcam; p-ERK (Thr202/Tyr204) #4370, ERK #9102, Cell Signaling), at a 1:2,000 dilution overnight at 4 °C. The next day, the membranes were washed three times with TBST and then incubated with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature for 1 h. Finally, positive labeling of the proteins on the membranes was visualized by enhanced chemiluminescence (ECL) using a Bio-Rad ECL kit (Solarbio).
8
Western Blot Analysis of Signaling Proteins
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The detailed procedure for western blotting has been described [44 (link)]. Western blotting used primary antibodies (diluted 1:1000) against the following proteins: AKT (sc-8312), phosphorylated AKT (sc-16646-R), ERK (sc-94), phosphorylated ERK (sc-7383), and vimentin (sc-6260); each antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against HIF-1α (Cell Signaling) and E-cadherin (cat. 610182, BD Biosciences, Franklin Lakes, NJ) were also used. Proteins separated by SDS-PAGE were transferred to a Hybond-C Extra membrane (GE Healthcare, Little Chalfont, UK) that was then subjected to western blotting with an appropriate primary antibody. Anti-mouse or anti-rabbit IgG conjugated to horseradish peroxidase was used as the secondary antibody for detection using an ECL western blot detection system (Millipore, Bedford, MA, USA), and band intensities were quantified by densitometry (Digital Protein DNA Imagineware, Huntington Station, NY).
9
Characterization of EMT Markers
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YAP antibody (#14074) was from Cell Signaling Technology. SOX9 (#AB5535) antibody was from Millipore. Antibodies against E-cadherin (sc-8426) and vimentin (sc-6260) were purchased from Santa Cruz Biotechnology. Anti-CD44 (EP44) and anti-vimentin (OTI5D7) antibodies were from ZSGB-BIO. siRNA against human YAP and SOX9, mimics and antisense antagomirs of miR-506-3p, and scramble control RNA oligos were purchased from GenePharma.
10
Investigating TGF-β Signaling Pathways
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The MMP2 (#40,994), phospho-SMAD2 (#3108), phospho-SMAD3 (#9520), SMAD2 (#3103), SMAD3 (#9523), SMAD4 (#38454), phospho-SMAD1/5/8 (#13820), SMAD1 (#6944), and ID2 (#3431) antibodies were obtained from Cell Signaling Technology. The α-tubulin (#sc-23948) and vimentin (#sc-6260) antibodies were obtained from Santa Cruz Biotechnology. The cytokeratin-7 antibody (#MAB3554) was obtained from Millipore. The HLA-G antibody (#11-499) was obtained from EXBIO. The recombinant human GDF-11 and BMP4 were obtained from R&D systems. The SB431542 was obtained from Sigma.
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