Anti twist1

The SK-MEL-5 cells (2 × 10⁵ cells/mL) were treated with PG or OBX at 500 nM for different time periods. Adherent and floating cells were collected, washed twice and lysed for 15 min at 4 °C in RIPA buffer (0.1% SDS, 1% NP-40, 0.5% sodium deoxycholate, 50 mM NaF, 40 mM β-glycerophosphate, 200 µM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride and complete mini-protease-inhibitor cocktail (Roche, Basel, Switzerland)). Protein concentration was determined with the BCA protein assay (Pierce, Rockford, IL, USA) and 50 µg of protein extracts were separated by SDS-PAGE and transferred to Immobilon-P membranes (Millipore, Bedford, MA, USA). Immunoblots were incubated with anti-twist1 (#AV37997, Sigma-Aldrich), anti-vimentin (#3932, Cell Signaling Technology, Danvers, MA, USA), anti-MMP1 (#IM35, Calbiochem, Merck KGaA, Billerica, MA, USA) or anti-actin (sc-1616, Santa Cruz Biotechnology, Dallas, TX, USA) antibodies, according to manufacturer’s instructions. Antibody binding was detected with secondary antibodies conjugated to horseradish peroxidase (Santa Cruz Biotechnology) and the ECL detection kit (Amersham, Buckinghamshire, UK). Actin was used as a gel-loading control. Representative results of independent experiments are shown.

We extracted the protein (including total, nuclear and cytoplasmic protein) of the cells using RIPA lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1%Triton X-100, and 1 protease inhibitor cocktail tablet/10 ml) and detected the protein concentration with a BCA kit (Beyotime, China). The western blotting was conducted as previously described [23 (link)]. The primary antibodies were anti-E-cadherin (Bioss, USA), anti-N-cadherin (Santa Cruz, USA), anti-Vimentin (CST, USA), anti-GSK-3-β (Bioss, USA), anti-β-Catenin (CST, USA), anti-Twist1 (Sigma, USA), anti-GAPDH (Santa Cruz, USA), and anti-Lamin B (Bioss, USA).