Anti sappα

Hippocampal tissues from 9-month-old APP/PS1 transgenic mice were homogenized in radioimmunoprecipitation assay buffer (RIPA buffer) and protease inhibitor cocktail. Also, cells obtained from different treatments were lysed in RIPA buffer containing a protease inhibitor cocktail. The following antibodies were used: anti-APP (1:1000, Sigma), anti-NgR (1:1000, Millipore), anti-sAPPα (1:100, IBL), anti-sAPPβ (1:500, Covance), anti-β-site APP cleaving enzyme 1 (BACE1) (1:1000, abcam), anti-β-CTF and anti-α-CTF (1:1000, Sigma), anti-ionized calcium-binding adapter molecule 1 (Iba1) (1:1000, WAKO), glial fibrillary acidic protein (GFAP) (1:1000, DAKO) or anti-β actin antibody (1:3000, Abcam), anti-RhoA (1:1000, Sigma), and anti-ROCK2 (1:1000, Sigma). The membranes were incubated with secondary goat anti-rabbit and mouse IgG (1:5000, Thermo) and electrochemiluminescence (ECL, Millipore) reagent. The band signals were detected using BIO-RAD (Hercules, CA, USA) gel analysis software.

Protein isolation and immunoblotting procedures were as described previously [22 (link)]. For quantification of sAPP fragments, differentiated cells were treated in 10 ml Optimem (Gibco Life Technologies, Paisley, UK), and media were concentrated (30 kDa Amicon Ultra 15 ml filter) by centrifugation (4,000 g) and subjected to SDS-PAGE with amounts presented relative to total protein. Primary antibodies used were: anti-sAPPβ (Covance, Alnwick, UK; 1:1,000), anti-sAPPα (IBL International, Hamburg, Germany; 1:50), anti-APP (Ab54, GlaxoSmithKline; 1:4,000), anti-phospho-PKB (protein kinase B; Ser⁴⁷³), anti-PKB and anti-HKII (hexokinase II) (Cell Signaling, Hitchin, UK; 1:1,000), anti-GLUT1 (Millipore, Nottingham, UK; 1:1,000) and anti-GLUT4 (Abcam, Cambridge, UK; 1:1,000). Glut1 (also known as Slc2a1), Glut4 (also known as Slc2a4) and HkII (also known as Hk2) mRNA was determined by TaqMan RT-PCR (Applied Biosystems, Paisley, UK; Prism Model 7700) using commercial primers and probe sets.

Levodopa, bromocriptine, carbidopa, L-685,458, TAPI-1, Y-27632, and Forskolin (FSK) were purchased from Selleck Chemicals (Houston, TX, USA). Piribedil was from Tocris Bioscience (Bristol, UK). β-Secretase Inhibitor IV (BSI IV) was from Calbiochem (Hayward, CA, USA). Recombinant human BDNF, GNDF, IGF-I were from Peprotech (Rocky Hill, NJ, USA). cAMP and L-Ascorbic acid were from Sigma (St Louis, MO, USA). CellTiter-Glo was from Promega (Madison, WI, USA) and Effectene Transfection Reagent was purchased from QIAGEN (Hilden, Germany). Immunoblotting was performed with the following antibodies: anti-PS1 N-terminus (1–65) (PRB-354P, Covance, Davis, CA, USA); anti-APH1aL C-terminus (245–265) (PRB-550P, Covance); anti-NCT (N1660, Sigma); anti-Pen2 (P5622, Sigma); anti-BACE1 N-termimus (AP7774b, Abgent, Suzhou, China); anti-APP-CTF (A8717, Sigma); anti-sAPPα (secreted Amyloid Precursor Protein-α) (11088, IBL, Hokkaido, Japan); anti-HA (H6908, Sigma); anti-actin (A2066, Sigma); anti-D₂R (sc-9913, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Immunostaining was performed with the following antibodies: anti-nestin (MAB353, Milipore, Darmstadt, Germany); anti-Sox2 (sc-17320, Santa Cruz); anti-Ki67 (556003, BD Transduction Laboratories, San Jose, CA, USA), and anti-Doublecortin (DCX) (sc-8066, Santa Cruz).