Western Blot Analysis of EMT Markers

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)].

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Lu M.Y., Liao Y.W., Chen P.Y., Hsieh P.L., Fang C.Y., Wu C.Y., Yen M.L., Peng B.Y., Wang D.P., Cheng H.C., Wu C.Z., Shih Y.H., Wang D.J., Yu C.C, & Tsai L.L. (2017). Targeting LncRNA HOTAIR suppresses cancer stemness and metastasis in oral carcinomas stem cells through modulation of EMT. Oncotarget, 8(58), 98542-98552.

Publication 2017

BCA protein assay kit PVDF membrane E-cadherin vimentin SNAIL1 TWIST GAPDH ECL-plus chemiluminescence substrate LAS-1000 plus Luminescent Image Analyzer

Antibodies Assay Buffer Cadherin Cells Chemiluminescence Gapdh Luminescent Membrane Np 40 Protein Pvdf Sds page Vimentin

Corresponding Organization :

Other organizations : Chung Shan Medical University, Chung Shan Medical University Hospital, Taipei Medical University, Taipei Municipal YangMing Hospital, Taipei Medical University Hospital

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Variable analysis

independent variables

Cell lysis using NP-40 buffer

dependent variables

Protein concentration determined using BCA protein assay kit
Protein expression levels of E-cadherin, vimentin, FN-1, SNAIL1, TWIST, and ZEB1 measured by Western blot

control variables

Protein samples separated by 10% SDS-PAGE
Proteins wet-transferred to PVDF membrane
Primary antibodies used: E-cadherin (1:500), vimentin (1:500), FN-1 (1:500), SNAIL1 (1:500), TWIST (1:500), ZEB1 (1:500), GAPDH (1:5000)
Corresponding secondary antibodies used
Immunoreactive bands developed using ECL-plus chemiluminescence substrate
Bands captured by LAS-1000 plus Luminescent Image Analyzer

positive controls

GAPDH as a loading control

negative controls

Not specified

Annotations

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