P gsk3 ser9

This assay was performed as described previously [14 ]. Primary antibodies used in this study are: ER (6F11) from Abcam, Cambridge, MA, USA; PR (sc-7208) and BCL2 (sc-509) from Santa Cruz Biotechnology, Santa Cruz, CA, USA; P-AKT-Thr308 (#2214) and P-AKT-Ser473 (#2118) from Epitomics, Burlingame, CA, USA; P-PRAS40-Thr246 (#2997), P-GSK3-Ser9 (#9323), P-ERK1/2-Thr202/Tyr204 (#9101), P-S6-Ser240/244 (#2211), P-4EBP1-Thr37/46 (#9459), P-mTOR-Ser2448 (#2971), AKT (#9272), ERK1/2 (#9102), GSK-3β (#9832), PTEN (#9559), β-actin (#4970), and c-PARP (#9541) from Cell Signaling Technology, Danvers, MA, USA. All our shown Western blotting images are from the same gel with the same exposure to allow for a complete comparison between lines and across treatments.

MDA-MB-231 cells were grown in DMEM, and 67NR, 4T07, and 4T1 breast cancer cell lines were grown in RPMI supplemented with 10% (vol/vol) FBS, penicillin (100 units/ml), and streptomycin (100 μg/ml; Invitrogen, Rockville, MD). The four human breast cancer cell lines MCF10A1 (M-I), MCF10AT1k.cl2 (M-II), MCF10CA1h (M-III), and MCF10CA1a.cl1 (M-IV) were obtained from Dr. Anita Roberts (NCI/NIH, Bethesda, MD). M-I, M-II, M-III, and M-IV cells were grown in DMEM/F12 (Invitrogen, Carlsbad, CA) containing 5% horse serum (Invitrogen) at 37°C with 5% CO₂. M-I and M-II cells were supplemented additionally with 10 μg/ml insulin (Sigma, St. Louis, MO), 20 ng/ml epidermal growth factor (Sigma), 0.5 μg/ml hydrocortisone (Sigma), and 100 ng/ml cholera toxin (Sigma). Antibodies specific for APP (22C11) were purchased from EMD Millipore; APP (4G8) from Covance. Specific antibodies for p27(C-19) and p21 (F-5) were from Santacruz and anti-β-actin (AC-15) was from Sigma. Antibodies purchased from Cell Signaling were AKT (#9772), pAKT Thr308 (#4056), pAKT Ser473 (#9271), pFOXO1 Thr24 (#9464), pGSK3 Ser9 (#9336), pp65 Ser536 (#3033), pERK1/2 (#9101), β-Catenin (#9562), PARP (#9542), and cleaved Caspase-3 (#9661). Anti-survivin antibody (AB8228) was purchased from Abcam. The anti-CD44 antibody (#15675-1-AP) was from Proteintech group and anti-GSK3b (KAP-ST002E) antibody was from Stressgen.

HL-60 and NB4 cells were maintained in RMPI1640 (GIBCO, Carlsbad, CA) supplemented with 10% (v/v) fetal bovine serum (FBS) (GIBCO), 1% penicillin and streptomycin (GIBCO). Huh7 cells were cultured in DMEM (GIBCO) supplemented with 10% (v/v) fetal bovine serum (FBS), 1% penicillin and streptomycin. Antibodies against cleaved caspase-3, Akt, p-Akt (Ser473), GSK-3β, p-GSK-3 (Ser9) and β-catenin were purchased from Cell Signaling Technology (Danvers, MA). Antibodies against GAPDH, SIRPα, and secondary antibodies against mouse or rabbit IgGs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Alexa Flour 488 (goat anti-rabbit IgG, green) and annexin V-PI apoptosis kit were purchased from Invitrogen (Carlsbad, CA). Arsenic trioxide (As₂O₃; ATO) was purchased from Sigma Aldrich (St Louis, MO). Cells in our experiment were treated with ATO at 5 μM concentration where indicated. Lithium chloride (LiCl) and SB-216763 were purchased from Sigma Aldrich. 48 hours post-infection of lentivirus, cells were treated with 5 μm or 10 μm LiCl, 5 mM or 10 mM SB-216763 for 4 h and then lysed for westert blotting analysis..