Anti ps9 gsk 3β

Total proteins were extracted using 1X cell lysing buffer (Cell Signaling Technology). Samples were separated by electrophoresis in SDS- polyacrylamide gels (12%) and transferred to PVDF membranes. Membranes were blocked for 1 h with PBST (PBS plus 0.1% Tween-20) containing 5% non-fat milk. Blots were incubated overnight at 4°C with primary antibodies in PBST containing 5% BSA at the manufacturer's recommended dilution. The antibodies were purchased from the following suppliers: FKBP4 (ProteinTech); anti-pS473 Akt, anti-pT308 Akt, anti-Akt, anti-pT286 Cyclin D1, anti-Cyclin D1, anti-E2F-1, anti-pT37/46 4E-BP1, anti-pS9 GSK-3β, anti-GSK-3β, and anti-mTOR (Cell Signaling Technology); anti-GAPDH and anti-Actin (Santa Cruz Biotechnology); anti-Tubulin and anti-PIK3R2 (Sigma-Aldrich). After washing, blots were incubated with either an anti-rabbit HRP, or an anti-mouse HRP-conjugated antibody (Cell Signaling Technology) for 1 h at 25°C. After washing, blots were developed with Supersignal WEST Pico Plus (Life Technologies SAS).

pCI/β-catenin and its mutants tagged with HA at N-terminus were constructed by PCR amplification from pcDNA3/β-catenin and confirmed by DNA sequence analysis. pGL4-1.0, pRL-TK (thymidine kinase promoter driven Renilla reniformis luciferase) and dual luciferase assay kit were bought from Promega (Madison, WI, USA). Luciferase driven by different length of or mutated promoter of human PME-1 in pGL4.10 were constructed by using human blood genomic DNA, purified by Spin Column Blood Genomic DNA Purification Kit (Sangon Biotech, China) as template and confirmed by sequencing. Monoclonal anti-PME-1, polyclonal anti-GAPDH, monoclonal anti-demethylated-PP2Ac (DM-PP2Ac), siGSK-3β, siGSK-3α and siβ-catenin were bought from Santa Cruz (Santa Cruz, CA, USA). Polyclonal anti-β-catenin, anti-Non-pS-β-catenin (dephosphorylated at S33/S37/T41), anti-GSK-3β and anti-pS9-GSK-3β were purchased from Cell Signaling Technology (Danvers, MA, USA). Monoclonal anti-HA was from Sigma (St. Louis, MO, USA). Horseradish peroxidase-conjugated anti-mouse and anti-rabbit IgG were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). ECL (enhanced chemiluminescence) kit was from Thermo Fisher Scientific (Rockford, IL, USA).