Anti human slug

Following antibodies were used in this study: Anti-human MELK antibody (in-house, previously described [14 (link)]), anti-human DEPDC1 (in-house, previously described [36 (link)]), anti-human FOXM1, anti-human p53 (Santa Cruz Biotechnology), anti-human Ki67 (Millipore), anti-human p21, anti-human Slug, anti-human Snail (Cell Signaling Technology), anti-human E-cadherin (BD biosciences), anti-HA (Roche), anti-FLAG M2, and anti-beta-actin antibodies (Sigma). OTS167 was dissolved in DMSO (Sigma) for cell culture or in 5% glucose solution for animal study. The target sequences of oligo siRNAs were 5′-CUUACGCUGAGUACUUCGAUU-3′ for MELK and 5′-AGUUCAUUGGAACUACCAAUU-3′ for Luciferase (control). DEPDC1 oligo siRNA was purchased from Santa Cruz Biotechnology (sc-78918).

Total protein from cells was lysed using M-PER Mammalian Protein Extraction Reagent (Thermo) supplemented with a protease inhibitor cocktail (Sigma, St Louis, MO, USA). Samples were denatured, and equal amounts of protein were subjected to SDS-PAGE, and then transferred to nitrocellulose membrane. After blocking with 5% non-fat milk in TBST for 60 min, membranes were incubated with primary antibody dissolved in 5% bovine serum albumin in TBST overnight at 4°C. The following primary antibodies were used: anti-human- E-cadherin (1:2000, 24E10; Cell Signaling Technology, Danvers, MA, USA), anti-human-Vimentin (1:2000, D21H3; Cell Signaling Technology), anti-human-Snail (1:1000, C15D3; Cell Signaling Technology), anti-human-Twist (1:1000; Cell Signaling Technology), anti-human-Zeb (1:1000, D80D3; Cell Signaling Technology), and anti-human-Slug (1:1000, C19G7; Cell Signaling). Human GAPDH (1:5000; KangChen, Shanghai, China) was used as an internal reference.