Sb216763

Glycogen synthase kinase 3-beta (GSK3b) kinase inhibitor (SB216763) was purchased from Sigma-Aldrich. To determine the phosphorylation status of NDRG1, immunoprecipitation was used to pull down the NDRG1 protein, followed by an in vitro phosphorylation assay, similarly as previously described [57 (link),58 (link),59 (link)]. Briefly, Protein A/G PLUS-agarose beads (Santa Cruz Biotechnology, Dallas, TX, USA) with an anti-GFP antibody were used to precipitate NDRG1 proteins. After elution of the immunoprecipitated complex, the protein solution was subjected to an in vitro kinase assay by incubation with recombinant GSK3b protein (Sigma-Aldrich, St. Louis, MO, USA) in a solution containing ATP. Phosphorylated proteins were subjected to SDS-PAGE analysis, and the phosphorylation status of NDRG1 was determined by hybridization with an anti-phosphoserine antibody (Abcam, Cambridge, UK). Alternatively, NDRG1-FL- or NDRG1-Dc-transfected cells were treated with GSK3b-specific inhibitor (SB21673) at various doses for 24 h. Cellular proteins were extracted and subjected to a pulldown assay with an anti-GFP antibody. The phosphorylation status of NDRG1 was revealed by phospho-serine-specific immunoblotting.

Human embryonic kidney 293 T with SV40 T antigen (HEK293T) cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich, Madrid, Spain) supplemented with 10% fetal bovine serum (Invitrogen, CA, USA) and 80 µg/ml gentamycin (Gibco, MA, USA). Human neuroblastoma cells (SH-SY5Y) were cultured in RPMI supplemented with 10% fetal bovine serum (Invitrogen) and 80 µg/ml gentamicin. The SH-SY5Y-α-SYN Tet-Off cells, described previously [37 (link), 38 (link)], were cultured in RPMI with 10% fetal bovine serum, 250 µg/ml G418 (Gibco), 50 µg/ml hygromycin B (Invitrogen), and 2 µg/ml doxycycline (DOX) (Sigma-Aldrich). The expression of α-SYN was switched on by DOX removal. Transient transfection of HEK293T cells was performed with TransFectin Lipid Reagent (Bio-RAD, CA, USA). The inhibitors SB216763, LY294002, and MG132 were from Sigma-Aldrich. Cycloheximide (CHX) was from Boehringer Mannheim (Stuttgart, Germany).

Cell culture media, Dulbecco’s Modified Eagle medium (DMEM) was purchased from Gibco Inc. (NY, USA). LPS (Escherichia coli 055:B5) and 3-(4,5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide (MTT) were purchased from Sigma–Aldrich (St. Louis, MO, USA). SB203580, PD98059 were purchased from Calbiochem (San Diego, CA). SB216763 and N-Acetyl Cysteine (NAC) were purchased from Sigma–Aldrich. Antibodies against iNOS, ATF3 and cyclin D1 were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA, USA). Other antibodies against IκB-a, p65, ERK1/2, phospho-ERK1/2 (Thr202/Tyr204) and b-actin were purchased from Cell Signaling (Bervely, MA, USA). All chemicals were purchased from Sigma-Aldrich, unless otherwise specified.

The AKT inhibitor LY294002, GSK3β inhibitor SB216763, NFκB inhibitor Bay 11–7082, FITC-dextran, and Evans blue were purchased from Sigma (Sigma-Aldrich, St. Louis, MO, United States). JNK agonist anisomycin was purchased from Beyotime (Beyotime Biotechnology, Shanghai, China). TNF-α was obtained from R&D Systems (Minneapolis, MN, United States). The following primary antibodies were applied in this study: anti-p-GSK3β, anti-GSK3β, anti-p-JNK, anti-JNK, anti-p-NFκB and anti-NFκB antibodies purchased from Cell Signaling Technology (Danvers, MA, United States); anti-FGF20, anti-VE-cadherin, anti-Claudin-5, and anti-GFAP antibodies obtained from Abcam (Cambridge, MA, United States); and anti-p-AKT, anti-AKT and anti-Occludin antibodies purchased from Invitrogen (Carlsbad, CA, United States) and Santa Cruz Biotechnology (Dallas, TX, United States), respectively. The secondary antibodies used in this study were goat anti-rabbit immunoglobulin G (IgG) H&L (HRP) and goat anti-mouse IgG-HRP purchased from Abcam (Cambridge, MA, United States) and Santa Cruz Biotechnology (Dallas, TX, United States), respectively.

Immortalized embryonic kidney cell lines, 293T/17 and HEK293, were purchased from American Type Culture Collection, ATCC (Manassas, VA, USA). The U937, U937-Mock, and U937-NDRG2 cell lines [26 (link)] were cultured in Roswell Park Memorial Institute (RPMI) -1640 medium containing 10% fetal bovine serum (FBS) (ATCC, Manassas, VA, USA), HEPES (Lonza, Basel, Swiss), β-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA), and penicillin and streptomycin (Lonza, Basel, Swiss). The 293T/17 and HEK293 cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% FBS, penicillin, and streptomycin at 37 °C in 5% CO₂. In some experiments, the cells were treated with chemicals As₂O₃, MG132, 2’,7’-dichlorofluorescin diacetate (DCFH-DA), okadaic acid, SB216763 (Sigma-Aldrich, MO, USA), and z-VAD-fmk (ENZO, Seoul, Korea). The shMcl-1 clones (TRCN0000005514 and TRCN0000005516) were purchased from Sigma-Aldrich.