Anti phospho gsk3β ser9

RES, 3-(4, 5-dimethylthiazol-2-yl)−2, 5-diphenyltetrazolium bromide (MTT), rabbit polyclonal anti-TH antibody, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 1-methyl-4-phenylpyridinium ion (MPP⁺) and Selegiline were all obtained from Sigma-Aldrich (St. Louis, USA). Hydroxypropyl methylcellulose (HPMC) with a viscosity of 50 cps was kindly supplied by Colorcon Co., Ltd. (Shanghai, China). MDCK and SH-SY5Y cells were provided by Guangzhou Jenniobio Biotechnology Co., Ltd. (Guangzhou, China). Anti-rabbit IgG, anti-Akt, anti-phospho-Akt (Ser473), anti-Gsk3β and antiphospho-Gsk3β (Ser9) antibodies were purchased from Cell Signaling Technology (Danvers, USA).

The following antibodies were used: anti-GAPDH (1:5000) and anti-CDKL5 (1:500) (Sigma); anti-phosphorylated Erk1/2 (1:1000), anti Erk1/2 (1:1000), anti-phospho-AKT-Ser⁴⁷³ (1:1000), anti-phospho-AKT-Thr³⁰⁸ (1:1000), anti-AKT (1:1000), anti-phospho-GSK-3β-Ser⁹ (1:1000), anti-GSK-3β (1:1000), anti-phospho-CRMP2-Thr⁵¹⁴ (1:1000) and anti-CRMP2 (1:1000) (Cell Signaling Technology); anti β-catenin (1:1000; BD Transduction Laboratories); anti-phospho-CREB-Ser¹³³ (1:1000) and anti-CREB (1:1000) (Upstate Biotechnology). For the preparation of total cell extracts, cells were lysed in RIPA buffer (Tris–HCl 50 mM, NaCl 150 mM, Triton X-100 1%, sodium deoxycholate 0.5%, SDS 0.1%, protease and phosphatase inhibitors cocktails 1%; Sigma). For the preparation of tissue extracts, the hippocampi from P19 mice were homogenized in RIPA buffer. Extracts were immediately processed by Western blot or kept frozen (− 80 °C) until assayed. Sample protein concentration was estimated using the Lowry method (Lowry et al., 1951 (link)). Equivalent amounts (50 μg) of protein were subjected to electrophoresis on a 10% SDS-polyacrylamide gel. Densitometric analysis of digitized images was performed using Scion Image software (Scion Corporation, Frederick, MD, USA) and intensity for each band was normalized to the intensity of the respective total protein levels or GAPDH band.

Asiatic acid and LY294002 were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). The anti-Akt and anti-phosphor-Akt antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA); the anti-GSK-3β and anti-phospho-GSK-3β (Ser9) antibodies were from Cell Signaling Technology, Inc. (Danvers, MA, USA); the anti-GLUT4 and anti-PPARγ antibody was from Boster Biological Technology, Ltd. (Wuhan, China); and the anti-glyceraldehyde phosphate dehydrogenase (GAPDH) antibody was obtained from KangChen Bio-tech Inc. (Shanghai, China).

Antibodies for IF studies were rat anti-alpha tubulin monoclonal antibody (Serotec, Kidlington, UK), rabbit anti-β-catenin antibody (Sigma-Aldrich) and secondary antibodies (Alexa Fluor-conjugated reagents obtained from Molecular Probes, Paisley, UK). For the identification of focal adhesions, a staining kit (Millipore, Nottingham, UK) was used. Actin filament and nuclear staining was performed with rhodamine-phalloidin (Molecular Probes, Life Technologies) and DAPI (Molecular Probes, Life Technologies) at recommended working concentrations. For western blotting, proteins (40 μg) were separated by SDS–PAGE and transferred to nitrocellulose. Antibodies used were anti-GSK-3β, anti-phospho-GSK-3β (Ser9) (Cell Signaling, Danvers, MA, USA), anti-GSK-3α+β (phospho Y279+Y216) (Abcam, Cambridge, UK) and secondary antibodies as per manufacturer instructions (Dako, Cambridgeshire, UK).