Acidic and basic cytokeratin ae1 ae3

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)].

For immunoblotting cells were grown on Matrigel and treated as represented in the image. In the case of GLT combination treatments, cells were pretreated with GLT and then with TGFβ1. Post 24 h of treatment, cells were trypsinized and washed with cold PBS to dissolve Matrigel. The cells were lysed in protease and phosphatase inhibitor (Thermo-Fisher) containing RIPA (Boston Bioproducts) buffer. The protein content of the lysate were quantified using BCA assay (Thermo Fisher Scientific) and western blot was performed with equal protein. Membranes were blocked for 1 h at room temperature (5% nonfat milk) followed by overnight incubation with primary antibody. Following primary antibodies were used – p Smad-2 (#18338), Smad-2 (#5339), SNAI1 (#3879), Cyclin D1 (#55506), (Cell signaling Technology), Fibronectin (#610077), N-Cad (#610921)(BD Biosciences), GAPDH (#G9295), and α tubulin (#T5201) (Sigma), Acidic and basic Cytokeratin AE1/AE3 (#MAB3412) (Millipore) and EMA (#GA62961) (Dako). Secondary antibodies (from sigma) were used at a concentration of 1:10,000. Equal loading was verified by immunoblotting with GAPDH or αTubulin.