Anti o glcnac

Acute hippocampal slices treated with 1 μM thiamet-G or DMSO vehicle in the recording aCSF for one hour at 37 °C were homogenized in a lysis buffer supplemented with protease and phosphatase inhibitors, and protein concentrations were determined by the Bradford protein assay. Laemmli sample buffer containing SDS was added to the protein samples, and boiled at 95 °C for 5 min. 20 μg proteins were loaded in each well, and separated by a 10% SDS-PAGE gel. The proteins were transferred to a PVDF membrane at 100 V for one hour, and the membrane was subsequently blocked with 5% skim milk in TBST (Tris-buffered saline, with 0.1% Tween 20) for one hour at room temperature. The membrane was then incubated with primary antibodies, anti-O-GlcNAc (Cat#: 9875 S, Cell Signaling, 1:1000) and anti-actin (Cat#: SC-8432, Santa Cruz, 1:1000), overnight at 4 °C. After being thoroughly washed with TBST, the membrane was incubated with a secondary antibody, anti-mouse IgG-HRP (Cat#: SC-2005, Santa Cruz, 1:5000), for one hour at room temperature. Protein bands were visualized and captured with the western chemiluminescent HRP substrate (WBKLS0100, Millipore) using the ImageQuant LAS 4000 system (GE Healthcare).

The following antibodies were used: Anti-Paucimannose (serum-free undiluted cell culture supernatant; clone: Mannitou, gift from B. Schmitz, Bonn, Germany); anti high-mannose (serum-free undiluted cell culture supernatant; clone 492, gift from B. Schmitz, Bonn, Germany); anti-polySia (1:10,000 dilution, clone 735 gift from R.Gerady-Schahn, Hannover Germany, IgG), anti-NCAM (1:1000 dilution clone 5B8 IgG Hybridoma Bank); anti-CML (1:10,000 dilution; clone CML56 abcam); anti-O-GlcNAc (1:10,000 dilution; clone CTD110.6, Cell Signaling); anti-Synapsin-1 (1:5000 dilution; polyclonal 64581 Abcam); anti-RAGE (1:1000 dilution; polyclonal 3611 Abcam).

For Western blot analysis, protein samples were separated by SDS/PAGE and transferred on to nitrocellulose membrane (Whatman). The membranes were blocked for 1 h in 5% non-fat milk powder or 5% BSA in TBS-0.2% Tween20 (TBS-T) buffer for 1 h at room temperature with gentle shaking. Then, the following primary antibodies, diluted in blocking buffer, were added to the membranes overnight at 4°C: anti-OGT (DM-17, Sigma–Aldrich), anti-OGT (Abcam ab177941), anti-O-GlcNAc (RL2, Abcam), anti-O-GlcNAc [C-terminal domain (CTD) 110.6, Cell Signaling], anti-TOM20 (F-10, Santa Cruz Biotechnology), anti-ATP synthase β subunit (ATPB, 3D5, Abcam), anti-Timm13 (A01, Abnova), anti-HSP60 (D307, Cell Signaling), anti-α-tubulin (12G10, Developmental Studies Hybridoma Bank, University of Iowa) and anti-α-tubulin (11H10, Cell Signaling). The following fluorescent secondary antibodies, diluted in 1% milk in TBS-T, were added to the membranes for 1 h at room temperature: donkey anti-rabbit 800, donkey anti-mouse 680, goat anti-rabbit 800 and goat anti-mouse 680 (LI-COR). Images were acquired and analysed using the LI-COR Odyssey Image System.