Whole-cell protein (20–50 μg) was initially separated using 8% SDS-PAGE and subsequently, transferred onto Nitran membrane (Sigma-Aldrich, USA). Next, the membranes were blocked with BSA (5%), and incubated separately with anti-DOC2B (1:5000; Proteintech, USA), anti-CTNNB1 and CDH1 (1:3000) (Developmental Studies Hybridoma Bank, University of Iowa, USA), p-ELK-1(Ser383), total ELK-1, p-AKT (Ser473), total AKT, p-ERK1/2(Thr202/Tyr204), total- ERK1/2 (Thr202/Tyr204), p-p38MAPK (Thr180/Tyr 182), total- p38MAPK, MacroH2A1.2, Tri methyl H3 lys9, SNAI1, SNAI2, ZEB1 and β-actin (1:3000, Cell Signaling Technologies, USA), CCNE (HE12), GSK3α/β, CDKN2A, CDKN1A, CDKN1B (1:3000, Santa Cruz Technologies, USA) TWIST2 (1:2000, Abcam, Cambridge, USA) and CLDN1, CDH2 VIM (1:3000, Cloud Clone, USA) at 4 °C, and then with anti-mouse IgG-HRP or anti-rabbit IgG-HRP (1:5000) (Cell Signaling, USA) secondary antibodies. SuperSignal™ West Pico Chemiluminescent Substrate (Thermo Scientific, USA) was used for visualization of proteins in the membranes using Image Quant LAS 4000 (GE Healthcare, USA).
Twist2
Manufactured by Abcam
Sourced in United States
TWIST2 is a protein that plays a role in the regulation of gene expression. It functions as a transcription factor, binding to DNA and influencing the transcription of target genes. TWIST2 is involved in various cellular processes, including embryonic development and cell differentiation.
Automatically generated - may contain errors
Lab products found in correlation
P akt, by Cell Signaling Technology (2 mentions)
Las 4000, by Cytiva (2 mentions)
Gapdh, by Santa Cruz Biotechnology (2 mentions)
Imagequant las 4000, by GE Healthcare (1 mentions)
Anti mouse igg hrp, by Cell Signaling Technology (1 mentions)
P erk1 2, by Cell Signaling Technology (1 mentions)
Total erk1 2, by Cell Signaling Technology (1 mentions)
Snai1, by Cell Signaling Technology (1 mentions)
Total p38 mapk, by Cell Signaling Technology (1 mentions)
P p38 mapk, by Cell Signaling Technology (1 mentions)
Macroh2a1, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Anti rabbit igg hrp, by Cell Signaling Technology (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Snai2, by Cell Signaling Technology (1 mentions)
Nitran membrane, by Merck Group (1 mentions)
P elk 1, by Cell Signaling Technology (1 mentions)
Twist1, by Abcam (1 mentions)
Snail1, by Abcam (1 mentions)
Snail2, by Abcam (1 mentions)
6 protocols using twist2
1
Protein Expression Analysis by Western Blot
2
Quantitative Analysis of RNA-Protein Interactions
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3
Protein Extraction and Western Blot Analysis
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Cells were harvested using the total protein extraction kit (KeyGEN BioTECH, Nanjing, China). Samples were incubated for 0.5 h on ice with agitation and then centrifuged at 14 000×g for 15 min. Protein concentrations were determined using the bicinchoninic acid (BCA) protein assay kit (Pierce). Protein samples were subjected to electrophoresis on SDS-polyacrylamide gradient gels, transferred to a PVDF membrane, and blocked in 5% non-fat milk in TBST (phosphorylated proteins were blocked in 5% BSA in TBST) for 2 h at room temperature. Blots were incubated with primary antibodies to the following proteins: ezrin (Abcam), cortactin (Abcam), E-cadherin (CST), α-SMA (Abcam), Slug (CST), Snail (Abcam), Twist (Abcam), Twist2 (Abcam), phosphorylated ezrin at Y-567 (CST), and GAPDH (Beyotime). GAPDH was used on the same membrane as a loading control. The signal was detected after incubation with anti-rabbit or anti-mouse IgG secondary antibody (Bioworld) coupled to peroxidase, using ECL (Millipore). Protein expression levels were evaluated by densitometric analysis.
4
Western Blot Analysis of Protein Expression
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Cells were lysed with RIPA buffer supplemented with protease inhibitor cocktails, quantified using Bradford method and 30–50 μg of protein was resolved on 10%–12% SDS-PAGE. Further proteins were transferred onto 0.45 μm nitrocellulose membrane (Bio-Rad, United States) and blocked in room temperature with 5% BSA (HiMedia, India) solution for an hour and incubated with respective primary antibodies [FOXM1 (1:2000) (Cell Signaling Technologies, United States); Caspase-9 (1:3000) (Cell Signaling Technologies, United States), B-Actin (1:5000) (Invitrogen, United States); SnaiI, CDH2, VIM (1:3000) (Cloud Clone, United States); TWIST2 (1:2000) (Abcam, United States); γH2AX (1:2000) (Novus Biologicals, United States) and RAD51 (1:2000) (a kind gift from Dr. Ganesh Nagaraju, Indian Institute of Science, Bangalore, India) (Santa Cruz Technologies, United States)] at 4°C overnight. Blots were then incubated with secondary antibodies, either anti-rabbit IgG (1:5000) or anti-mouse IgG (1:5000) (Cell Signaling Technologies, United States) conjugated with HRP for an hour at room temperature. Finally chemiluminescent signals were developed using Clarity Western ECL substrate (Bio-Rad, United States) and visualized using ImageQuant LAS 4000 instrument (GE Healthcare, United States).
5
Comprehensive Protein Extraction and Western Blot Analysis
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Protein was extracted with RIPA buffer (Thermo Fisher) supplemented with protease inhibitor cocktails I and II (Sigma) and phosphatase inhibitor cocktail set III (Calbiochem). Protein was quantified with Pierce BCA protein assay kit (Thermo Fisher) and analysed with a Sunrise Tecan plate reader. After Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and electro‐transfer onto Polyvinyl‐Difluor membranes (GE Healthcare), Western blotting was performed with the following primary antibodies and concentrations: GAPDH (sc‐365062; Santa Cruz; 1:10,000), PAX3 (AB_528426; DSHB; 0.5 μg/mL), Twist2 (ab66031; Abcam; 1 μg/mL), IMP1/2/3 (sc‐271785; Santa Cruz; 1:1000), HMGA2 (no. 5269S; Cell Signalling; 1:1000), tubulin (ab210797; Abcam; 1:1000), p53 (sc‐126; Santa Cruz; 1:100), citrate synthase (G‐3; sc‐390693; Santa Cruz; 1:1000). Blots were stained with secondary antibodies conjugated with Horseradish Peroxidase, 1:2500, (Promega W401B, W402B) and visualized with ECL prime western blotting detection reagent kit (GE Healthcare).
6
Western Blot Analysis of Skeletal Muscle
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Skeletal muscle lysate was diluted in Laemmli buffer solution and separated by SDS-PAGE. After transfer to nitrocellulose membranes (Bio-Rad), proteins were blocked in 7.5% nonfat milk, washed with TBST (10 mmol/l Tris-HCl, 100 mmol/l NaCl, and 0.02% Tween 20), and incubated with primary antibody overnight at 4 8C. The membranes were washed with TBST and incubated with appropriate HRP-conjugated secondary antibodies (Bio-Rad, diluted 1:25 000). Proteins were visualized by ECL (GE Healthcare, Little Chalfont, UK) and quantified using Quantity One Software (Bio-Rad). Primary antibody for glycogen synthase (GS) (catalogue item: #3893), GS phosphorylated on serine 641 (p-GS) (#3891), ACC (#3676), ACC phosphorylated on serine 79 (p-ACC) (#3661), AKT (or protein kinase B (PKB)) (#9272), and its phosphorylated form on serine 473 (p-AKT) (#9271) were purchased from Cell Signaling (Danvers, MA, USA), GAPDH (#sc-25778) was purchased from Santa-Cruz Biotechnology and TWIST2 (#ab66031) from Abcam (Cambridge, UK). Additional TWIST1 antibodies from Santa-Cruz Biotechnology (#sc-15393 and #sc-6269) and Sigma-Aldrich (#T6451) were also used.
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