Ecl plus western blot detecting system

Cell proteins were extracted from cells using RIPA buffer (Cell Signaling, Danvers, MA) containing a phosphatase inhibitor (Roche, Indianapolis, IN). Equal amounts of protein were loaded and separated through SDS-PAGE. The proteins were then transferred to a membrane, and the blot was blocked with 10% non-fat milk in TBST. The membranes were probed overnight at 4°C using the following antibodies: PI3 Kinase p110α, p-4E-BP1 (T70), 4E-BP1 (53H11), phosphor-AKT (Ser473), Akt (pan), p-GSK-3-alpha/beta (S21/9) (D17D2), GSK-3alpha/beta (D75D3), p-Tuberin/TSC2 (Ser939), p-beta-catenin (Ser33/34/Thr41), LRP6 (C5C7), p-LRP6 (S1490) (Cell Signaling, Danvers, MA), Myc (9E10) (Santa Cruz Biotechnology, Dallas, TX), FZD7 and β-actin (Sigma-Aldrich, St. Louis, MO). Appropriate antibodies were used for secondary antibody reactions. The ECL Plus Western Blot Detecting System (GE Healthcare, Piscataway, NJ) was employed to detect the presence of antibodies.

Cell protein was extracted from cells using RIPA buffer (Cell Signaling, Danvers, MA) with phosphatase inhibitor (Roche, Indianapolis, IN). Equal amount of protein was loaded and separated by SDS-PAGE. After the protein was transferred onto a membrane, the blot was blocked with 5% non-fat milk in TBS and probed overnight at 4°C using the following antibodies: WNT5B (Sigma-Aldrich, St. Louis, MO), Cyclin E (M20), TOM20 (F-10), Myc (9E10), AIF (E-1), MCL1 (S-19) (Santa Cruz Biotechnology, Santa Cruz, CA), Caspsae-3, Caspase-8,PGC-α, Cyclin D1 (Cell Signaling, Danvers, MA) and β-actin (Bio-Rad, Hercules, CA). Appropriate antibodies were used for secondary antibody reaction. Signal was detected by the ECL Plus Western Blot Detecting System (GE Healthcare, Piscataway, NJ).

Cells were seeded in 6-well plates and lysed in ice-cold RIPA buffer, which contained phosphatase and protease inhibitors cocktail (Basel, Roche). After centrifugation at 12000 rpm/min for 15 min at 4°C, the concentration of proteins in the supernatants was detected with BCA Protein Assay Kit (Beyotime, Shanghai, China). Equal amounst of proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE), transferred onto nitrocellulose (NC) membranes and blocked with 5% non-fat milk in Tris buffered saline tween (TBST). Membranes were incubated overnight at 4°C with the following primary antibodies: PDIA6, Cyclin D1, Cyclin A1, Cyclin B1, Cyclin E1, c-myc, β-Catenin (ProteinTech, Wuhan, China), CK1α, GSK-3β, phosphor-GSK-3β (Ser9), β-TrCP (Abcam, Cambridge, MA, USA); phosphor-β-Catenin (Ser45), phosphor-β-Catenin (Ser33/Ser37/Thr41, Cell Signaling Technology, Danvers, MA, USA); β-actin (ZSGB-BIO, Beijing, China) and Histone H3 (Beyotime). After washing, the membranes were incubated with secondary antibodies (HRP-conjugated anti-rabbit IgG or anti-mouse IgG, ProteinTech) for 2 h at RT. Bands were visualized using ECL Plus Western Blot Detecting System (GE Healthcare, Beijing, China).