For western blot, samples were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and blotted with various antibodies; anti-GAPDH (#2118), N-cadherin (#13116), E-cadherin (#3195), Vimentin (#5741), Slug (#9585), Snail-1 (#3879), ZEB1 (#3396), ZO-1 (#8193), phospho-Met (Tyr1349) (#3121), Ron (#4269), pβ-catenin (Thr41 or Ser45) (#9565), β-catenin (#8480), S6 (#2217), total Akt (#9272), phospho-c-Myc (Thr58/Ser62) (#9401), Ki-67 (#9027), phospho-AKT (Ser473) (#9271), phospho-EGFR (Tyr1068) (#11862), LRIG1 (#12752), c-Met (#8198) and antibodies were purchased from Cell Signaling Technology. LRIG1 (G-20) (#sc-50075) and EGFR (#sc-03) antibodies were purchased from Santa Cruz Biotechnology. c-Myc antibody (clone 9E11) (#MS-127-P0) was purchased from Neomarkers. Fibronectin (#GTX112794), CD44 (#GTX102111) and Twist (#GTX127310) were purchased from Genetex (CA, USA). Actin (#A5441) and Tubulin (#T5168) were purchased from Sigma (MO, USA). All antibodies used horseradish peroxidase-conjugated secondary antibodies (Biorad), followed by developing with SuperSignal West chemicals (Pierce). An AlphaInnotech imaging station with FluorChem software was used to capture images. All data are representative of more than three independent experiments.
Twist
Manufactured by GeneTex
Sourced in United States
The Twist is a versatile laboratory instrument designed for a range of applications. It features precise temperature control and rotational mixing capabilities, making it suitable for a variety of sample processing tasks.
Automatically generated - may contain errors
Lab products found in correlation
Vimentin, by GeneTex (4 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
N cadherin, by Cell Signaling Technology (3 mentions)
Vimentin, by Cell Signaling Technology (3 mentions)
Snail1, by Cell Signaling Technology (3 mentions)
E cadherin, by GeneTex (3 mentions)
N cadherin, by GeneTex (3 mentions)
Total akt, by Cell Signaling Technology (2 mentions)
β catenin, by Cell Signaling Technology (2 mentions)
Anti gapdh, by Cell Signaling Technology (2 mentions)
E cadherin, by Cell Signaling Technology (2 mentions)
Phospho met tyr1349, by Cell Signaling Technology (2 mentions)
P β catenin, by Cell Signaling Technology (2 mentions)
Phospho c myc thr58 ser62, by Cell Signaling Technology (2 mentions)
Phospho akt ser473, by Cell Signaling Technology (2 mentions)
Phospho egfr tyr1068, by Cell Signaling Technology (2 mentions)
Lrig1, by Cell Signaling Technology (2 mentions)
C met, by Cell Signaling Technology (2 mentions)
Lrig1 g 20, by Santa Cruz Biotechnology (2 mentions)
Horseradish peroxidase conjugated secondary antibody, by Bio-Rad (2 mentions)
11 protocols using twist
1
Comprehensive Western Blot Analyses
2
Western Blot Analysis of EMT Markers
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After transfection for 48 h, cells were lysed using RIPA buffer (Thermo, USA) with proteinase inhibitor cocktail (Roche), then incubated at 4 °C for 15 min. The lysate was centrifuged and protein concentration was measured by BCA Protein Assay Kit (Thermo, USA). The protein was separated on 10% sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to 0.22 μm PVDF membrane (Thermo, USA). After blocking with 5% fat-free milk for 1 h, the membranes were incubated with primary antibodies at 4 °C overnight with gentle shaking. Next, the membranes were incubated with secondary antibody at room temperature for 2 h. Finally, the immunofluorescence of protein bands was visualized using Pierce™ ECL Western Blotting Substrate (32,109, Thermo Scientific, USA) by Image J 2 software (Madison, WI, USA). Primary antibodies are as following: E-cadherin (1: 500, ab15148, Abcam company), Vimentin (1:500, #7431, Cell Signaling Technology (CST)), GAPDH (1:5000, 60,004-l-lg, Proteintech company), Rac1 (1:500, ab33186, Abcam), Twist (1500, GTX60776, Gene Tex).
3
2D-DIGE Analysis of Galectin-1 Signaling
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Fluorescent dyes (Cy2, Cy3, and Cy5) and reagents for 2D-DIGE were purchased from GE Healthcare (Uppsala, Sweden). Lipofectamine® RNAiMAX Transfection Reagent and OPTI-MEM were purchased from Invitrogen (Waltham, MA, USA). MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was purchased from USB Corp. (Cleveland, OH, USA). Propidium iodide (PI) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Recombinant human LGALS1 protein (rhLGALS1) was purchased from BioLegend, Inc. (San Diego, CA, USA). LGALS1, cyclin A2, cdk2, phosphor-ERK (Thr202), MMP-9, MMP-3, ZEB2, SNAI1, TWIST, E-cadherin, N-cadherin, and vimentin primary antibodies were purchased from Genetex Inc. (Hsinchu, Taiwan). Phospho-p38 MAPK (Thr180/Tyr182) and p38 MAPK primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Cyclin D2, cdk4, cyclin E, and p27 primary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). ERK1/2 primary antibody was purchased from Promega Corp. (Madison, WI, USA). Anti-rabbit and anti-mouse immunoglobulin (Ig)G secondary antibodies were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA, USA). All the chemicals and reagents used in this study were of analytic grade.
4
Western Blot Analysis of EMT Markers
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Cells were lysed in NP-40 buffer and protein concentration was determined using BCA protein assay kit (Thermo Fisher Scientific, Rockford, IL, USA). Samples were separated by 10% SDS-PAGE and wet-transferred to a PVDF membrane (Millipore, Billerca, MA, USA) followed by incubation with primary antibodies against E-cadherin (Santa Cruz. Biotechnology Inc., Santa Cruz, CA, USA; 1:500), vimentin (Santa Cruz; 1: 500), FN-1 (Santa Cruz; 1: 500), SNAIL1 (Cell Signaling Technology Inc., Danver, MA, USA; 1: 500), TWIST (GeneTex, Irvine, CA, USA; 1: 500), ZEB1(Santa Cruz; 1: 500) or GAPDH (GeneTex, Irvine, CA, USA; 1: 5000). After incubation with corresponding secondary antibodies, the immunoreactive bands were developed using an ECL-plus chemiluminescence substrate (Perkin-Elmer, Waltham, MA, USA) and captured by LAS-1000 plus Luminescent Image Analyzer (GE Healthcare, Piscataway, NJ, USA) [55 (link)].
5
Twist-Induced Protein Profiling
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Twist (GeneTex); p50, p65, Hsp27, Smurf2, Ubiquitin, and β-actin (Santa Cruz Biotechnology); phospho-Hsp27 (Ser78), phospho-Hsp27 (Ser82), phospho-IkBα (Ser32/36), and Lamin A/C, Flag (Cell Signaling Technology); IkBα, IL6, IL1β, pro-SPC, and phospho-Hsp27 (Ser15) (Abcam); phospho-Hsp27 (Ser86) (Thermo Fisher Scientific); α-SMA and Flag (Sigma); and Alexa488-conjugated phalloidin (Invitrogen). The detailed antibodies for immunoblotting, immunohistochemical and immunofluorescence staining were provided in Additional file 1 : Table S2.
6
Western Blot Analysis of EMT Markers
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The protein lysate was subjected to SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane. Protein expression was analyzed by western blotting using primary antibodies against E-cadherin (Clone 36B5; NeoMarkers, Fremont, CA, USA), Vimentin (C-20; Santa Cruz Biotechnology, Santa Cruz, CA, USA), Slug (Santa Cruz Biotechnology), Twist (GeneTex), Annexin A2 (GeneTex), phosphorylated and total Akt (Cell Signaling Technology, Beverly, MA, USA), phosphorylated and total Erk (Cell Signaling Technology), or β-actin (Sigma-Aldrich), followed by incubation with horseradish peroxidase-conjugated secondary antibodies. Specific proteins were detected by chemiluminescence using ECL Plus Western Blotting Detection Reagents (GE Healthcare Biosciences, Piscataway, NJ, USA).
7
Erlotinib and Quercetin Pathway Analysis
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Quercetin and other chemicals were purchased from Sigma (St. Louis, MO, USA) unless specified otherwise. Erlotinib (Tarceva, OSI Pharmaceuticals, Melville, NY, USA) was purchased from Lumtec (Hsinchu, Taiwan), antibodies against Akt, E-cadherin, GAPDH, p-Akt, and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and anti-p-EGFR antibodies were purchased from Millipore (Burlington, MA, USA). Anti-EGFR, ERK, HK2, LDHA, MMP-2, PKM2, p27, Twist, and Vimentin antibodies were purchased from GeneTex, Inc. (Irvine, CA, USA). Antibodies against GLUT1, p-ERK, N-cadherin, and α-tubulin were obtained from Cell Signaling Technology (Danvers, MA, USA). MMP-9 and p21 antibodies were obtained from Abcan (Cambridge, UK) and Proteintech (Rosemont, IL, USA), respectively. PKM2 siRNA and the Lipofectamine RNAiMAX reagent were purchased from Invitrogen (Grand Island, NY, USA).
8
Comprehensive Western Blot Analyses
9
Western Blot Analysis of EMT Markers
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Cell lysates were collected using radioimmunoprecipitation assay (RIPA) buffer supplemented with a protease and phosphatase inhibitor cocktail (Roche, Mannheim, German). Approximately 30~50 μg of proteins was separated in denaturing sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). After blocking with 1% bovine serum albumin/TBST blocking buffer, the membranes were incubated overnight with specific primary antibodies. Membranes were washed three times with the TBST wash buffer and probed with an appropriate horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA). Protein bands were detected with an enhanced chemiluminescence (ECL) reagent (Millipore). A primary antibody for YBX1 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies for α-tubulin, E-cadherin, vimentin, Twist, Slug, and N-cadherin were purchased from GeneTex (San Antonio, TX, USA). Western blotting was performed at least three times, and representative experiments are shown.
10
Proteomic Analysis of Cell Lysates
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For total lysate preparation, cells were lysed in RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% sodium dodecyl sulfate, and 1% Nonidet P-40 (NP-40); pH 7.4) supplemented with proteinase inhibitors (complete proteinase inhibitors, Roche, Indianapolis, IN, USA). Protein lysates (30 μg per lane) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and processed for immunoblotting with antibodies against cleaved PARP (Enogene, Atlanta, GA, USA, E11-0365L), CD44 (GeneTex, Irvine, CA, USA, CTX102111), ALDH1 (GeneTex, GTX123973), Nanog (GeneTex, GTX100863), Oct4 (GeneTex, GTX101497), Bmi1 (GeneTex, GTX114008), Snail (Abcam, Cambridge, UK, Ab78105), Twist (GeneTex, GTX127310), FoxM1 (GeneTex, GTX100276), and Tubulin (Enogene, E1C601), respectively. Signals were detected using an enhanced chemiluminescence system (ECL, NEN Life Science, Boston, MA, USA).
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