Nuclear Protein Expression Analysis

Cells were treated for 96 h with 2 mM VPA, and at the end of the treatment, equal concentrations of cell lysate from nuclear fractions were obtained as already described [38 (link)], were electrophoresed on a 4–12% NuPAGE Novex Bis-Tris gel (Life Technologies by Thermo Fisher Scientific, Waltham, MA, USA) with MES (NuPage Novex, Life Technologies by Thermo Fisher Scientific, Waltham, MA, USA). Western blots were performed using standard procedures [38 (link)]. Primary antibodies used were: goat anti-Snail1 (1:1000, AbCam, Cambridge, UK), and rabbit anti-Twist1 (1:500, Santa Cruz Biotechnology, Dallas, TX, USA). As loading control protein, rabbit anti-Histone 3 (1:2000, Sigma-Aldrich) was used. Proper HRP-conjugated secondary antibodies were used (1:2000 anti-rabbit, Bio-Rad, Hercules, CA, USA, 1:5000 anti-goat Santa Cruz Biotechnology). Proteins were visualised using SuperSignal West Pico or Dura chemiluminescent substrate (Thermo Fisher Scientific, Waltham, MA, USA) with a Kodak Image Station 440 CF (Eastman Kodak Co., New Haven, CT, USA). Bands were then quantified with ImageJ (https://imagej.nih.gov/ij/) and results were normalised versus controls. The experiments were performed at least in triplicates.