Optiblot ecl detect kit

Cells were seeded, cultured and exposed with different treatments for 72 h. Whole cell protein lysates were prepared according to standard protocol using RIPA buffer (0.5 M Tris–HCl, pH 7.4, 1.5 M NaCl, 2.5% deoxycholic acid, 10% NP-40, 10 mM EDTA). Protein (50 μg) was loaded per well of a 12% SDS PAGE gel using electrophoresis buffer (0.192 M glycine, 25 mM Tris, 0.1% SDS). After electrophoresis, the gel was transferred onto a PVDF membrane (Bio-Rad Laboratories, Inc., CA, USA) using transfer buffer (0.192 M glycine, 25 mM Tris, 0.025% SDS, 10% methanol). Membranes were blocked in TBS-T with 5% non-fat dry milk and incubated overnight with the primary anti-Bax (1:1000, Abcam), anti-Bcl-2 antibody (1:250, Abcam) or anti-cleaved-caspase-3 (1:1000, Cell signaling), then incubated with secondary HRP-linked antibody (1:5000) (KPL Inc., USA). Detection was done by Abcam Optiblot ECL Detect Kit (Abcam, USA). Anti-β-tubulin antibody (1:20000) (Sigma-Aldrich Co., St. Louis, MO, USA) was used to for loading correction.

Total cell protein lysates were extracted using RIPA buffer (0.5 M Tris–HCl, pH 7.4, 1.5 M NaCl, 2.5% deoxycholic acid, 10% NP-40, 10 mM EDTA) by cultured on ice for 10 min. Protein concentration was measured by performing BCA assay (Sigma-Aldrich, USA). 20 μg of total protein was fractionated using 6–12% gradient SDS-PAGE gel using electrophoresis buffer (0.192 M glycine, 25 mM Tris, 0.1% SDS). Fractionated blots were transferred onto a PVDF membrane (Millipore, USA), which was blocked in TBS-T with 5% non-fat dry milk and incubated overnight with the primary antibodies at dilution of 1:1000 as follows: Oct-4 (cat. No.: ab181557), CD44 (cat. No.:ab243894), Sox2 (cat. No.: ab93689), β-actin (cat. No.: ab8226), cleaved Poly [ADP-ribose] polymerase 1 (PARP1, cat. No.: ab32064), cleaved caspase-3 (cat. No.: ab32042), MetAP-2 (cat. No.: ab134124), p53 (cat. No.: ab26), p21 (cat. No.: ab109520). Then membrane was incubated with secondary HRP-linked antibody at dilution of 1:5000. Detection was done by Abcam OptiBlot ECL Detect Kit (Abcam, USA).

To investigate whether DKK1 exerts the same effect on the Wnt pathway in HepG2/C3A and PLC/PRF/5 cells, protein levels of active beta-catenin were determined by western blotting. MMP-2 and MMP-9 protein levels were also evaluated. Cells were lysed using RIPA protein extraction reagent (Sigma-Aldrich, USA) in the presence of sodium orthovanadate, a protease inhibitor cocktail and PMSF, all purchased from Sigma. The supernatant was collected and concentrated by ultracentrifugation. Protein concentrations were measured with a BCA protein-assay Kit (BIO-RAD, USA) for cell lysates and with the Bradford reagent (BIO-RAD, USA) for the concentrated supernatants. Equal amounts of protein samples were subjected to 10% SDS-PAGE analysis and electrophoretically transferred to PVDF membranes. Membranes were then blocked with 5% BSA (Sigma-Aldrich, USA) for 1 hr, then probed with primary antibodies: rabbit monoclonal antibody MMP-2 (40994), rabbit monoclonal antibody MMP-9 (13667), or rabbit monoclonal non-phospho (active) β-catenin (8814) at 4°C overnight, then incubated with HRP-linked anti-rabbit antibody at room temperature for 2 hrs. All antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Signals were visualized using an Optiblot ECL Detect Kit (Abcam, USA).