Gsk 3β

Iscove’s Modified Dulbecco’s Medium (IMDM), fetal bovine serum (FBS), Lipofectamine LTX, plus reagent, matrigel, DMEM/F12, B27 supplement, N2 supplement, and Anti-Neu2 were from Invitrogen (MA5-25555) (USA), as well as CD133 and CD44 (BD phermingen) and Oct-4A(2840), Sox2(3579), Nanog(4903), Slug(9585S), Snail(3879S), Cyclin D1(2978S), mTOR(2983), phospho-mTOR (Ser2481)(2974), Rictor(9476), GSK-3β(9315), Gli1(3538S), Sufu(2522S),GSK-3β(9315), Phospho-GSK-3β-Ser9(5558), Akt(4691), Phospho-Akt-Ser473(4060), β-Actin(4970), HRP-conjugated anti-rabbit antibodies(7047S) and anti-mouse secondary antibodies (7076). SignalStain^® DAB Substrate Kit(8059) was from Cell Signaling Technology (Danvers, USA). Shh(sc-9024) antibody was from Santa Cruz Biotechnology (Texas, USA). Basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), anti-Caspase3, active antibody(C8487) and other chemicals were from Sigma–Aldrich. RevertAid First Strand cDNA Synthesis Kit (K1622) and Maxima SYBR Green qPCR Master Mix (2X) were from Thermo Scientific, USA (K0251); biotinylated Sambucus nigra agglutinin (SNA)(B-1305) and biotinylated Maackia amurensis agglutinin (MALII) (B-1265) were from Vector Laboratories (USA).Rictor plasmid Addgene plasmid #11367) was from Addgene and PcDNA3.1-Neu2 was a kind gift from Dr. Eugenio Monti.

siRNA knockdown was performed in Opti-MEM media (Gibco) and Dharmafect 1 Transfection Reagent (Horizon Discovery). A non-specific siRNA (ON-TARGETplus; Horizon Discovery) was used as a control alongside siRNA against GSK3-β (Invitrogen). Knockdown in wells of a 6-well plate was performed by incubating 40 nM siRNA with Dharmafect 1 for 20 min before applying to cells. The next day, cell culture media was refreshed, and cells were grown for another 3 days before downstream experiments.

The cells were lysed in RIPA buffer (Cell signaling, USA). The protein extract were separated on SDS–polyacrylamide gels and were electrotransferred to a nitrocellulose membrane (GE healthcare life sciences, UK). The membranes were blocked in 5 % non-fat dry milk and probed with primary antibodies for SPHK-1 (Cell signaling, USA), HIF-1α (Novus Biologicals, USA), AKT (Santa Cruz Biotechnology, Santa Cruz, CA, USA), p-AKT (Santa Cruz Biotechnology, Santa Cruz, CA, USA), GSK-3β (Invitrogen, USA), p-GSK-3β (Cell signaling, USA), VEGF (Santa Cruz Biotechnology, Santa Cruz, CA, USA), PCNA (DAKO, USA), PI3K (Millipore, Germany), and β-actin (Sigma-Aldrich, St, Louis, MO, USA) overnight at 4 °C and HRP-conjugated secondary antibodies. Detection of specific proteins was carried out with an enhanced chemiluminescence (ECL) assay (GE Healthcare Life Sciences, UK).

Following various treatments, the cells were washed once in 1X PBS and lysed with RIPA buffer (150 mM NaCl, 1.0% Triton-X-100, 0.5% Sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0) with 1 mM PMSF. Protein concentrations were determined by Bradford Assay using Pierce 660 nm Protein Assay reagent (thermofisher, Waltham, MA, USA). Following SDS-PAGE, Proteins were transferred to nitrocellulose membranes, which were blocked for 1-hour in 5% milk with gentle agitation. Membranes were incubated overnight at 4°C with various antibodies then washed in TBS-T (20mM Tris, 150mM NaCl, 0.1% Tween 20), and incubated for 1-hour at room temperature with horseradish-peroxidase-linked anti-Mouse or anti-Rabbit IgGs (Cell Signaling, Danvers, MA, USA). Protein bands were detected by chemiluminescence. Antibodies: ROR1 (Cell signal, #D6T8C), GAPDH (Cell signal, #D16H11), CK1ε (Cell signal, #12448S), p-AKT-ser473 (Cell signal, #9271S), AKT (Cell signal, #C67E7), p-GSK3β-ser9 (Cell signal, #D17D2), GSK3β (Cell signal, #D7SD3), XIAP (Cell signal, #D2Z8UL), caspase-9 (Cell signal, #9502S), Bad (Cell signal, #D24A9), p-Bad-ser136 (Cell signal, #4366T), β-catenin (Invitrogen, MA1-301), p-β-catenin-ser33/37, Wint-5a (Novus-Bio NBP2-24752SS)

We used bead-based multiplex ELISAs to measure immunoreactivity to the
insulin receptor (IR), IGF-1 receptor (IGF-1R), IRS-1, Akt, proline-rich Akt
substrate of 40 kDa (PRAS40), ribosomal protein S6 kinase (p70S6K), and glycogen
synthase kinase 3β (GSK-3β), and ^{pYpY1162/1163}-IR,
^{pYpY1135/1136}-IGF-1R, ^pS312-IRS-1,
^pS473-Akt, ^pT246-PRAS40, ^pTpS421/424-p70S6K,
and ^pS9-GSK-3β (Invitrogen, Carlsbad, CA). Samples (100
μg protein) were incubated with the beads, and captured antigens were
detected with secondary antibodies and phycoerythrin-conjugated anti-rabbit IgG
[33 (link)]. Plates were
read in a MAGPIX (Bio-Rad, Hercules, CA).

We used bead-based multiplex ELISAs to measure immunoreactivity to the insulin receptor (IR), IGF-1 receptor (IGF-1R), IRS-1, Akt, proline-rich Akt substrate of 40 kDa (PRAS40), ribosomal protein S6 kinase (p70S6K), and glycogen synthase kinase 3β (GSK-3β), and pYpY1162/1163-IR, pYpY1135/1136-IGF-1R, pS312-IRS-1, pS473-Akt, pT246-PRAS40, pTpS421/424-p70S6K, and pS9-GSK-3β (Invitrogen, Carlsbad, CA). Samples (50 μg protein) were incubated with the beads, and captured antigens were detected with secondary antibodies and phycoerythrin-conjugated anti-rabbit IgG. Plates were read in a MAGPIX (Bio-Rad, Hercules, CA).