Protein Expression Analysis by Western Blot

Cells were lysed on ice in lysis buffer [10 mM Tris-HCl (pH 7.4), 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM Na₄P₂O₇, 2 mM Na₃VO₄, 1% Triton X-100, 10% glycerol, 0.1% SDS, 0.5% deoxycholate] (Invitrogen, Camarillo, CA, USA) supplemented with 1 mM phenylmethylsulfonyl fluoride and protease inhibitor cocktail (Sigma, St. Louis, MO, USA). Whole cell extracts (20 µg/lane) were electrophoresed using 10% SDS-polyacrylamide gels and electrotransferred to polyvinylidene fluoride membranes (Millipore, Schwalbach, Germany) as described previously.3 (link) The following antibodies were used for Western blotting: anti-HIF-1α (1:1000), -E-cadherin (1:500), -N-cadherin (1:500), -vimentin (1:1000), -Twist (1:1000), -Slug (1:1000), and GAPDH (1:1000). All antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).