Immunoblotting Analysis of EMT and Autophagy

Immunoblotting was performed following protocols as described previously [46 (link)]. Cells were lysed in modified RIPA buffer (Sigma-Aldrich) and protein content was measured using Bradford reagent (Thermo Scientific). The cellular protein lysates were run in denaturing polyacrylamide gels and thereafter transferred to PVDF membrane (Thermo Fisher Scientific, #88518) for blocking with 5% skimmed milk (HiMedia). The blots were then probed or re-probed with specific primary antibodies and detected using enhanced chemi-luminescence (ECL; Thermo Fisher Scientific, #3210) detection system following the manufacturer’s protocol. The primary antibodies used were as follows: anti-E-cadherin (CST, 24E10), N-cadherin (CST, D4R1H), Vimentin (CST, D21H3), β-catenin (CST, D10A8), ATG5 (CST, D1G9), LC3-II (CST, D11), phospho-total Smad-2/3 (CST) and Smad-7 (Santacruz, #sc-365846). The secondary antibodies were horseradish peroxide-conjugated goat anti-rabbit IgG. Expression was quantitated using ImageJ and analyzed through Graph-pad Prism software [47 (link)].