Chemi genius

Whole-cell lysates were prepared in 2× Laemmli sample buffer. Proteins were separated by Sodium dodecyl sulphate-polyacrylamide gel electrophoresis, on 7% gels (26 ). The proteins were transferred to a PVDF membrane in a wet transfer cell (Bio-Rad) using a buffer containing 25 mM Trizma base and 192 mM Glycine. After transfer the proteins were fixed to the membrane in 100% methanol for 5 min. The membranes were blocked in 5% non-fat milk before probing with the relevant antibody: γ-tubulin, diluted 1:10 000 (Sigma-Aldrich T6557), RB1 1:500 (BD Biosciences 554136), RB1-phospho (ser795) 1:1000 (Abcam ab47474). Detection was performed using an enhanced chemiluminescence kit (Amersham Biosciences) and quantified in a Chemi Genius (Syngene) with the software GeneTools.

Equal quantities of cell extract were loaded onto a 10% SDS-polyacrylamide gel and analyzed using the Western lightning plus-ECL detection system (PerkinElmer, Inc, Waltham, MA, USA). Blotting membranes were probed using GDF15 antiserum (A0185; Abclonal, Cambridge, MA, USA), MASPIN antiserum (554292; BD Biosciences), NDRG1 antiserum (42-6200; Invitrogen), NDRG2 antiserum (ab169775; Abcam, Cambridge, MA, USA), NDRG3 antiserum (ab131266; Abcam), E-cadherin antiserum (1.B.54; Santa Cruz Biotechnology), N-cadherin antiserum (AJ1526a; Abgent, San Diego, CA, USA), SLUG antiserum (C19G7; Cell signaling, Danvers, MA, USA), SNAIL antiserum (ab117866; Abcam), or β-actin antiserum (I-19, Santa Cruz Biotechnology). Band intensities were recorded using the Chemi Genius II BioImaging System of Syngene (Cambridge, UK) and analyzed using the GeneTool Program of ChemiGenius (Syngene).