Western Blot Analysis of EMT Markers

Total cell lysate was prepared in RIPA (Boston Bioproducts) containing protease and phosphatase inhibitor cocktail (Thermo). The cell lysate was quantified and proteins separated on SDS-PAGE and transferred to the PVDF (Bio-Rad) membrane. Membranes were blocked in 5% nonfat milk in TBS with 0.1% Tween-20 (TBST) for 1 h at room temperature followed by incubation with primary antibodies in TBST with 1% BSA overnight. The following primary antibodies were used – p Smad-2, p Smad-3, Smad-2, Smad-3, Snail, Vimentin, EPCAM, c-Myc, NFAT-1, Cyclin B, Cyclin D1, (Cell signaling Technology), Fibronectin, N-Cad (BD Biosciences), Pancytokeratin, GAPDH, α tubulin and β actin (Sigma), Acidic and basic Cytokeratin AE1/AE3 (Millipore), EMA (Dako), p27 (Santa Cruz Biotechnology), and cytokeratin-8 (Developmental Studies Hybridoma Bank, University of Iowa). Secondary antibodies (from sigma) were used at a concentration of 1:10,000. Equal loading was verified by immunoblotting with GAPDH, αTubulin or β Actin [44 (link)].

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Dhar Dwivedi S.K., McMeekin S.D., Slaughter K, & Bhattacharya R. (2015). Role of TGF-β signaling in uterine carcinosarcoma. Oncotarget, 6(16), 14646-14655.

Publication 2015

RIPA protease and phosphatase inhibitor cocktail PVDF Vimentin EPCAM c-Myc NFAT-1 Cyclin B Cyclin D1 Fibronectin N-Cad Pancytokeratin GAPDH α tubulin and β actin Acidic and basic Cytokeratin AE1/AE3 cytokeratin-8

Acidic Actin Antibodies Basic cytokeratin C myc Cell Cyclin b Cyclin d1 Cytokeratin 8 Epcam Fibronectin Gapdh Hybridoma Membranes Milk Phosphatase Protease Proteins Pvdf Ripa Sds page Smad 3 Snail Tween 20 Vimentin α tubulin

Corresponding Organization :

Other organizations : University of Oklahoma Health Sciences Center

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Variable analysis

independent variables

Cell lysate preparation method (RIPA buffer with protease and phosphatase inhibitors)
Antibody incubation conditions (primary antibodies incubated in TBST with 1% BSA overnight)

dependent variables

Protein expression levels of p Smad-2, p Smad-3, Smad-2, Smad-3, Snail, Vimentin, EPCAM, c-Myc, NFAT-1, Cyclin B, Cyclin D1, Fibronectin, N-Cad, Pancytokeratin, GAPDH, α tubulin, β actin, Acidic and basic Cytokeratin AE1/AE3, EMA, p27, and cytokeratin-8

control variables

Blocking conditions (5% nonfat milk in TBST for 1 h at room temperature)
Secondary antibody concentration (1:10,000)
Equal loading verification (GAPDH, αTubulin or β Actin)

controls

Positive controls: Not explicitly mentioned
Negative controls: Not explicitly mentioned

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