Anti transgelin

Cultured cells were lysed in RIPA buffer [150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS) and 50 mM Tris buffer, pH 8.0]. The proteins were heated at 100°C for 10 min and analyzed by SDS-polyacrylamide gel electrophoresis on 8–12% polyacrylamide gels. The proteins were electroblotted onto PVDF membranes (Millipore, Milford, MA, USA). The membrane blots were blocked with 5% (w/v) non-fat dried milk, incubated with primary and secondary antibodies, and then visualized with the enhanced chemiluminescence western blotting detection system (WEST-ZOL plus; iNtRON Biotechnology, Daejeon, Korea). Anti-transgelin was purchased from Abcam, while the anti-extracellular regulated kinase (ERK)1/2 and anti-phosphorylated ERK1/2 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-α-tubulin, HRP-conjugated goat anti-rabbit IgG and HRP-conjugated goat anti-mouse IgG antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Protein lysates were harvested from cells at 48 hours after transfection, washed with phosphate-buffered saline (PBS), and lysed on ice in lysis buffer (1% NP-40, 50 mM Tris–HCl pH 8.0, 150 mM NaCl and 2 mM EDTA) supplemented with complete protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN, USA). The lysates were centrifuged at 15,000 rpm for 30 min at 4°C. The supernatant was collected. Protein concentration was quantitated by BCA method. About 30–50 μg of protein were separated by SDS−PAGE and then transferred to PVDF membranes. The membranes were probed with primary antibody for 3 h. After incubation with HRP-conjugated antibody, antibody detection was achieved by chemiluminescence. The primary antibodies used in this study were: as follow anti-transgelin-2 (Novus Biologicals), anti-transgelin (Abcam, Cambridge, MA, USA), anti-AKT (Santa Cruz Biotechnology), anti-phospho-AKT (Santa Cruz Biotechnology) and anti-GAPDH (Cell Signaling Technology, Danvers, MA, USA).

The nuclear and plasma proteins from HCT116, SW480, LOVO and RKO cell lines were extracted using the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, USA). The protein concentration of the extracted cytoplasmic and nuclear proteins was determined. Immunoblotting was performed with the primary antibody anti-transgelin (1:500, Abcam, USA, or 1:500, R&D, USA), anti-GADPH (1:400, Abcam, USA or 1:500, Cell signaling technology, USA), anti-PARP1 (1:500, Cell signaling technology, USA), anti-Lamin B1(1:1000, Cell signaling technology, USA), anti-flag (1:500, Cell signaling technology, USA) and the secondary antibody (horseradish peroxidase (HRP)-conjugated goat anti-rabbit or anti-mouse IgG, 1:30,000, Sigma-Aldrich, USA) or IgG Detector (IgG Detector Solution v2, HRP labeled, 1:1000, Takara, Japan). Antibody detection was performed using a chemiluminescence substrate and the protein bands were visualized with Syngene G: BOX Chemi XT4 fluorescence and chemiluminescence gel imaging system (Cambridge, UK).