Anti app

Aggrecan from bovine articular cartilage (cat no. A1960), and poly-L-ornithine hydrobromide (Mw 30,000–70,000; cat no. P3655) were purchased from Sigma-Aldrich. Recombinant human pleiotrophin produced in yeast was described previously [37 (link), 41 (link), 60 (link), 61 (link)]. Anti-PTPRZ-S, rabbit polyclonal antibodies against the extracellular region of PTPRZ [62 (link)], and rabbit polyclonal antibodies against phosphorylated Tyr-1105 of p190 RhoGAP [43 (link)] were described previously. The following are the sources of commercially available regents and antibodies used in the present study: Anti-aggrecan (clone Cat-315; cat no. MAB1581, Millipore), anti-MBP (cat no. sc-13914, Santa Cruz Biotechnology), anti-NG2 proteoglycan (cat no. AB5320, Millipore), anti-phosphotyrosine (clone PY20; cat no. ab16389, Abcam), anti-p190 RhoGAP (cat no. 610150, BD Biosciences; and cat no. 12164, Cell Signaling Technology), anti-FYN (cat no. P2992, Sigma-Aldrich; and cat no. 4023, Cell Signaling Technology), anti-phosphorylated Tyr-416 of Src (cat no. 2101, Cell Signaling Technology), anti-phosphorylated Tyr-527 of Src (cat no. 2105, Cell Signaling Technology), anti-GFAP (cat no. Z0334, Dako), anti-OLIG2 (cat no. AB9610, Millipore), and anti-APP (cat no. ab15272, Abcam).

After 24 hours of treatment with A1 or vehicle, the M17 cells were harvested in 2 x SDS-PAGE sample buffer. The samples were resolved on 10% SDS-PAGE gels and transferred to PVDF membranes. Then, the membranes were blocked in 5% milk for one hour, followed by incubation overnight at 4°C with the corresponding primary antibody. The primary antibody was anti-APP, which is specific to the APP amino terminal (Abcam, Cambridge, UK), and anti-BACE1 (Sigma Aldrich, St. Louis, MO, USA). On the subsequent day, the membranes were washed three times with TBST buffer for five minutes. The membranes were then incubated in 5% milk (v/v) supplemented with anti-rabbit IgG or anti-mouse IgG for one hour at room temperature. Following incubation with the secondary antibody, the membranes were washed three times with TBST buffer for five minutes. The blots were then visualized via incubation with a SuperSignal West Dura chemiluminescence kit (Pierce Biotechnology, Rockford, IL, USA). Images were obtained via an AlphaImager HP System (Alpha Innotech, San Leandro, CA, USA). β-actin was used as a reference control for protein loading. The bands were quantified using Fluorchem Q SA software (Alpha Innotech, Santa Clara, CA, USA).

Proteins were isolated from the hippocampus of brain according to the protein extraction kit instructions (Beyotime Biotechnology, China). Protein concentrations were determined using the BCA protein assay kit (Bioworld, USA). Equal volumes of protein were separated using 10% dodecyl sulfate, sodium salt polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were blocked in 5% non-fat milk for 1 h and incubated overnight at 4 °C with primary antibodies as follows: 6E10 (1:1000; Covance, USA), anti-APP (1:1000, Abcam Technology, UK), anti-PSD95 (1:1000, Cell Signaling Technology, USA), anti-SYN (1:1000, Abcam Technology, UK), anti-IDE (1:1000,Abcam Technology, UK), anti-ADAM10 (1:1000, Millipore Technology, USA), anti-BACE1 (1:1000, Millipore Technology, USA), anti-PS1 (1:1000, Cell Signaling Technology, USA), and anti-GRPDH (1:2000, Cell Signaling Technology, USA). Membranes were rinsed with Tris-buffered saline with Tween 20 (TBST) three times. Then, membranes were incubated with HRP-conjugated anti-rabbit or anti-mouse secondary antibodies for 2 h. Protein signals were visualized with the chemiluminescence reagents provided with the ECL kit (Bioworld, USA), and quantitation of proteins was determined by densitometric analysis using ImageJ software (Bio-Rad, Hercules, USA).

All chemicals and reagents used here were purchased from Sigma (St. Louis, MO, U.S.A.) unless otherwise indicated. Anti-APP, anti-BACE1, anti-sAPPα, and anti-sAPPβ antibodies were from Abcam (Cambridge, U.K.). Anti-COX-2, anti-iNOS, anti-p-p65, anti-p-65, anti-p-IκBα, anti-IκBα, anti-IKK-α (IκB kinase α), anti-NF-κB1 p105, anti-NF-κB1 p50, and anti-β-actin antibodies were from Cell Signaling Technology (Danvers, MA, U.S.A.).

Equal amounts of proteins (50 μg) were loaded on 10% SDS-PAGE and transferred onto polyvinylidenedifluoride membranes. The membranes were blocked for 1 h with a blocking buffer containing 5% BSA in Tris-buffered saline solution and Tween 20 (10 mM Tris-HCl, 150 mM NaCl, 0.05% Tween 20; TBS-T). Membranes were then incubated overnight at 4°C with different primary antibodies diluted in the same blocking buffer. Incubations with HRP-conjugated secondary antibodies (Sigma) were performed for 1 h at room temperature and visualized by quantitative chemiluminescence using ECL Western blotting detection reagents (Millipore). Signal intensity was quantified using Image J. Antibodies used were as follows: anti-APP (1:1,000, Abcam), anti-Aβ (1:1,500, Abcam), anti-NICD (1:1,000, Abcam), and anti-Hes 1 (1:1,000; Santa Cruz Biotechnology). To control for loading, blots were stripped and reprobed with mouse anti β-actin (1:2,000; Santa Cruz Biotechnology; Smrt et al., 2010 (link)).

Cryosections of brain were fixed with 4% paraformaldehyde, penetrated with 1% Triton X-100, and then washed three times with phosphate-buffered saline (PBS). Sections were incubated with 10% donkey serum albumin (Abcam) in PBS for 30 min, after which primary antibodies were added and incubated at 4°C overnight. The following primary antibodies were used: mouse anti-Nestin (1:150, Abcam, Cambridge, MA, USA), rabbit anti-Sox2 (1:150, Abcam), anti-neuronal nuclei (NeuN, 1:150, Abcam), anti-galactocerebroside (GalC, 1:150, Chemicon), anti-glial fibrillary acidic protein (GFAP, 1:150, Abcam), anti-NF-M (1:150, StemCell Technologies, Vancouver, BC, Canada), anti-APP (1:150), anti-Aβ (1:150, Abcam) and anti-synaptophysin (SYP, 1:150, Abcam). After washing three times with PBS, sections were incubated with Cy3-conjugated donkey anti-rabbit immunoglobulin lgG secondary antibodies (1:200, Jackson, West Grove, PA, USA) for 1 h at room temperature. All of these were then supplemented with DAPI nuclear dye (1:100, Sigma), washed third with PBS, and viewed using an inverted fluorescence microscope (Gu et al., 2015 (link)). Image J (National Institutes of Health, Bethesda, MD, USA) was used for quantitative analysis.

The following antibodies were used in this study: anti-Fyn (catalogue no. ab1881, Abcam), anti-Fyn (catalog no.: sc-434, Santa Cruz Biotechnology), anti-APP (catalog no.: ab32136, Abcam), anti-actin (catalog no.: TA-09, Zsgb-Bio, China), anti-NMDAR2B (catalog no.: ab254356, Abcam), anti-ZDHHC21 (catalog no.: PA5-25,096, Invitrogen, USA), anti-NeuN (catalog no.: ab279295, Abcam), anti-tau (phospho T231; catalog no.: ab151559, Abcam), anti-tau (phospho Thr181; catalog no.: 701530, Invitrogen), anti-Aβ (catalog no.: ab201060, Abcam), anti-phospho-Src family (catalog no.: 2101S, Cell Signaling Technology, USA), horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG heavy chain-specific secondary antibody (catalog no.: A25222, Abbkine, China), HRP-conjugated mouse anti-rabbit IgG light chain (catalog no.: A25022, Abbkine), goat anti-mouse IgG H&L (HRP; catalog no.: ab6789, Abcam), goat anti-rabbit IgG H&L (HRP; catalog no.: ab205718, Abcam), goat anti-rabbit IgG H&L (Cy3; catalog no.: ab6939, Abcam), and goat anti-mouse IgG H&L (FITC; catalog no.: ab6785, Abcam).