Anti phospho gsk3β s9

We resolved cell proteins by SDS-PAGE before electro-blotting onto PVDF membranes. We incubated the membranes with the following primary antibodies overnight: anti-NCYM (1∶1000 dilution), anti-MYCN antibody (1∶1000 dilution; Calbiochem and Cell Signaling), anti-Lamin B (1∶1000 dilution; Calbiochem), anti-α-tubulin (1∶1000 dilution; Santa Cruz, CA, USA), anti-GST (1∶1000; Santa Cruz), anti-GSK3β (1∶1000 dilution; Cell Signaling), anti-phospho-GSK3β (S9) (1∶1000 dilution; Cell Signaling), anti-β-catenin (1∶1000 dilution; Cell Signaling), anti-phospho-AKT (S473) (1∶1000 dilution; Cell Signaling), anti-phospho-AKT (S308) (1∶1000 dilution; Cell Signaling), anti-AKT (1∶1000 dilution; Cell Signaling), anti-S6K (1∶1000 dilution; Cell Signaling), anti-phospho-S6K (T389) (1∶1000 dilution; Cell Signaling), and anti-actin (1∶4000 dilution; Sigma). The membranes were then incubated with a horseradish peroxidase-conjugated secondary antibody (anti-rabbit IgG at 1∶2000–1∶4000 dilution or anti-mouse IgG at 1∶2000 dilution; both from Cell Signaling Technology) and the bound proteins were visualized using a chemiluminescence-based detection kit (ECL and ECL pro kit, Amersham, Piscataway, NJ, USA; ImmunoStar LD, Wako).

Proteasomal inhibitor MG132 and GSK3β inhibitor SB415286 were purchased from Selleckchem.com and TOCRIS bioscience (Bristol, UK), respectively. Lithium chloride was obtained from Calbiochem (La Jolla, CA). Cycloheximide was purchased from Sigma (St. Louis, MO). Human and mouse Wnt3a was purchased from R & D Systems (Minneapolis, MN). Antibodies were purchased as follows: anti-CyclinD1, anti-HA, anti-c-Myc, anti-RhoA, and anti-poly (ADP) ribose polymerase (PARP) from Santa Cruz Biotechnology (Santa Cruz, CA); anti-STRAP and anti-β-catenin from BD Transduction Labs (San Jose, CA); anti-phospho-β-catenin (Ser33/37/Thr41), anti-GSK-3β, anti-APC and anti-phospho-GSK-3β (S9) from Cell Signaling Technology (Danvers, MA); and anti-β-actin and anti-Flag antibodies from Sigma (St. Louis, MO).

The following antibodies were purchased from Cell Signaling Technology: anti-phospho-tyrosine (p-Tyr-100), anti-Pim1, anti-Pim2, anti-Pim3, anti-Cyclin D3, anti-phospho-eIF4B, anti-AKT, anti-phospho-AKT (S473), anti-phospho-AKT (T308), anti-p70S6K, anti-phospho-p70S6K (T389), anti-phospho-S6 (S240/244), anti-phospho-GSK3β (S9), and anti-c-Myc, and anti-phospho IRS1 (S1101, S318, S302, S307, S636/639, S612) antibodies which recognize the following residues in human (h) or mouse (m) IRS1; anti-phospho IRS1 hS1101 (m1097); mS318 (hS323); mS302 (hS307); mS307 (hS312); hS636/S639 (mS632/635); mS612 (hS616). This numbering refers to human IRS1 (P35568); and mouse IRS1 (P35569). Anti-IRS1 (R & D systems), anti-RPS6 (Santa Cruz Biotechnology), anti-His (GenScript), anti-HA and anti-β-actin (Sigma) antibodies were used in these studies. Horseradish peroxidase (HRP)-linked enhanced chemiluminescence (ECL), mouse IgG and rabbit IgG secondary antibodies were purchased from GE Healthcare Life Sciences. Various protein kinase inhibitors, GNE-652 (Genentech), LGB321 (Novartis), AZD1208, AZD5363 (AstraZeneca) and PP242 (Selleck Chemicals) were used in these studies. All other chemicals including cycloheximide (CHX) and PMA were purchased from Sigma. Human recombinant insulin and insulin-like growth factor 1 (IGF1) were obtained from Peprotech.