β actin antiserum

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).

ASCs were washed with PBS and whole cell extracts were prepared by resuspending cell pellets in 100 μl lysis buffer (20 mM HEPES (pH 7.7) [Sigma-Aldrich], 300 mM NaCl [Sigma-Aldrich], 1.5 mM MgCl2 [Sigma-Aldrich], 0.2 mM EDTA [Sigma-Aldrich], 0.1% Triton X-100 [Sigma-Aldrich], 0.5 mM dithiothreitol [Sigma-Aldrich] and a cocktail of protease inhibitors [Roche]) and incubated at 4°C for 1 hour. The lysate was then centrifuged at 13,000g for 15 min at 4°C to remove cell debris. 50 mg of denaturated protein extract was resolved on a 10% polyacrylamide (Sigma-Aldrich) SDS-gel, transfered to a PVDF membrane (BioRad), and incubated with STAMP2 antiserum (Abcam, 1:1000) in TBS+0.1% Tween, or with GAPDH antiserum (Santa Cruz, 1:1000) or β-actin antiserum (Santa Cruz, 1:1000) in 5% BSA TBS+0.1% Tween (Sigma-Aldrich). Secondary antibodies used: horseradish peroxidise-conjugated (HRP) α-mouse IgG antibody (1:5000, Sigma), HRP α-rabbit IgG antibody (1:10000, Sigma). Immunoreactive bands were detected using ECL Western blotting reagents (GE Healthcare) and images were obtained using a film developer (Optimax, Protech).

Cells were incubated in the RPMI-1640 medium with 10% FCS and different treatments for a period of 24 hours. The nuclear and cytoplasmic fractions were extracted by NE-PER Nuclear and Cytoplasmic Extraction Reagent Kit (Thermo Scientific, Rockford, IL). Equal quantities of cell extract were loaded onto a 10% sodium dodecyl sulfate polyacrylamide (SDS) gel and analyzed by the electrochemiluminescent detection system. The blotting membranes were probed with 1∶1000 diluted IκB kinase α (IKKα) antiserum, 1∶1000 NFκBp50 antiserum, 1∶1000 diluted NFκBp65 antiserum (Merck Millipore, Darmstadt, Germany), 1∶1000 diluted PARP (Cell Signaling, Danvers, MA), 1∶200 diluted NFκB-inducing kinase (NIK) antiserum, 1∶1000 diluted IκB antiserum, 1∶200 diluted Lamin B antiserum, or 1∶3000 diluted β-actin antiserum (Santa Cruz Biotechnology, Santa Cruz, CA). The intensity of different bands was recorded and analyzed by GeneTools of ChemiGenius (Syngene, Cambridge, UK).