Anti glycogen synthase kinase 3 beta gsk3β

Five rats from each group were decapitated, and their brains removed and placed on ice plates. Bilateral SVZ were dissected and frozen in liquid nitrogen. Total protein was extracted in lysates containing protein inhibitor cocktail. Protein (30 μg) was fractionated on 10% sodium dodecyl sulfate-polyacrylamide gels for electrophoresis, and then transferred onto polyvinylidene fluoride membranes. Membranes were blotted overnight with anti-insulin receptor β (IRβ) (1:1,000), anti-glycogen synthase kinase 3 beta (GSK3β) (1:1,000; Cell Signaling, Boston, MA, USA), anti-phospho-glycogen synthase kinase 3 beta (p-GSK3β) (1:1,000; Cell Signaling), anti-β-catenin (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:5,000; Santa Cruz Biotechnology) at 4°C. Membranes were washed with Tris-buffered saline containing Tween 20 and then blotted with corresponding horseradish peroxidase-conjugated secondary antibodies (1:5,000). Blotted bands were visualized by enhanced chemiluminescence and analyzed by Image J software (NIH, Bethesda, MD, USA). All western blot experiments were performed at least three times. Lanes were scanned and optical density normalized using GAPDH as an internal control.

Human colon cancer HCT116 and LoVo cells were harvested after treatment, the total or NP40 protein was extracted, and protein expression was detected by Western blotting as previously described with minor modifications [35] (link). Equal amounts of proteins were size fractionated by 9% to 15% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. anti-SIRT6 (2590S, Cell Signaling, Danvers, MA), anti-PKCζ (sc-17781, Santa Cruz, CA), anti-PI3K (ab191606, abcam, Cambridge, MA), anti–serine/threonine kinase 1 (AKT) (9272, Cell Signaling, Danvers, MA),anti–glycogen synthase kinase-3 beta (GSK3β) (9315, Cell Signaling, Danvers, MA), anti–serine/threonine protein phosphatase 2A (PP2A) (ab3210, abcam, Cambridge, MA), anti-phospho-(Ser/Thr) (9631, Cell Signaling, Danvers, MA), anti-Flag (F1804, Sigma Aldrich), anti-His (PM032, MBL), anti-GST (sc-138, Santa Cruz, CA), anti-MYC (M047-3, MBL, Japan), anti–α-tubulin (BE0031, EASYBIO, Beijing, China), and anti–β-actin (4967, Cell Signaling, Danvers, MA) were used, and the blots were developed using an enhanced chemiluminescence kit (Amersham Corp.).