α β actin

For quantification of protein levels after miRNA expression, transfected MiaPaCa cells were fractionated using the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific, Braunschweig, Germany). 20 μg of protein from the nuclear fraction was loaded onto a 10% polyacrylamide gel and was then electrophoretically transferred to a nitrocellulose membrane. The membrane was blocked with Tween-20 (0.05%)-TBS (pH 7.4; 0.1 M Tris Base, 1.4 M NaCl) containing 5% milk, followed by incubation with respective primary antibody α-TCF4 (LS-Bio, Eching, Germany) at a concentration of 1:1000 or as a control α-β-Actin (Abcam, Cambridge, UK) with a concentration of 1:2000. Membranes were washed with Tween-20 (0.05%)-TBS and were incubated with a horseradish peroxidase (HRP)-conjugated anti-rabbit antibody (1:5000). Signals were detected using the enhanced chemiluminescence system (ECL, Amersham Life Science Ltd., Bucks, UK). Films were scanned with a CanoScan 9900F scanner (Canon, Japan). Protein levels were quantified using the Odyssey software LI-COR and normalized to the β-Actin control.

Cells were lysed with lysis buffer comprising 50 mM Tris pH 8.0, 150 mM NaCl supplemented with fresh 0.5% NP-40, 0.5 mM DTT, 1 × protease inhibitors cocktail (Roche, Switzerland) and 1.3 μl of Benzonase (Novagen, Germany) (1 hr, 4°C). Samples were prepared by boiling 40 μg of total protein extract with Laemmli buffer (Life Technologies, Cambridge, USA) . Samples were analysed using Bolt 10% Bis-Tris +SDS PAGE (Life Technologies, Cambridge, USA) and electroblotted onto 0.2 µm pore Whatman-Protran nitrocellulose membranes (Capitol Scientific, Austin, USA) in transfer buffer comprising 25 mM Tris/0.21M glycine/20% methanol. Membranes were blocked using 5% (w/v) low-fat milk in 0.01% (v/v) Tween-20/PBS (PBSTw) before incubating with primary antibody in blocking solution . Membranes were washed with PBSTw before incubating with donkey-raised secondary antibodies conjugated with IRDye 800CW (1:10000; LI-COR 926–32213, Lincoln, USA) and HRP-conjugated α-βActin (1:10000; Abcam ab20272, UK) antibody. HRP-staining was developed using a Super-signal West Pico kit (Pierce, Cambridge, USA) before imaging the membranes using LI-COR Odyssey Fc imager. The primary antibodies used were: α-Sox2 (1:1000; Abcam ab92494, UK) and α-Nanog (1:2000; Bethyl Laboratories A300-397A, Montgomery, USA).