Tnfr1

After drug treatment, cells extracts were collected in 2×SDS sample buffer and protein concentrations were determined with Amido Black Protein Assay. Equal amounts of total protein lysate (20 µg) were loaded onto SDS-PAGE gels for separation and transferred to nitrocellulose membrane for immunoblotting. The following primary antibodies were used: ph-NF-κB, IκBα, and cleaved caspase-3 (Cell Signaling Technology, Danvers, MA, USA), β-actin (CoWin Bioscience, Beijing, China), TNFR1 (Abcam, Cambridge, MA, USA), cadherin-11 (Life Technologies, Carlsbad, CA, USA). The appropriate anti-mouse Ig and peroxidase-conjugated anti-rabbit Ig secondary antibodies (CoWin Bioscience, Beijing, China) were used. The signals were detected and quantified using a Fluorchem FC2 Luminescent Imager (Alpha Innotech).

After the incubation ex vivo, slices were added to tubes containing ice-cold Krebs buffer with or without 2 mM BS3 (Rockford) and incubated for 30 min at 4°C. Cross-linking was terminated by quenching the reaction with 100 mM glycine during 10 min at 4°C. The slices were homogenized as above and analyzed by immunoblotting using the primary antibodies: CCR2 (1:1000, Novus), GAT3 (1:1000, Synaptic Systems), KCC2 (1:1000, Millipore), TNFR1 (1:500, Abcam) and TrkB (1:500, Abcam). Membrane expression was calculated as the difference between the intensity of the bands without BS3 (total protein) and with BS3 (non-membrane protein).

After the end of administration, the hippocampal tissues of the mice were collected before lysis in RIPA buffer on ice. The supernatant was acquired after centrifugation. Then protein concentration of the sample was determined using bicinchoninic acid (BCA) assay, which was followed by protein separation by sodium dodecyl sulfate-polyacrylamide denaturing gel electrophoresis (SDS-PAGE). The next step was transferring the separated proteins from the gel onto polyvinylidene fluoride (PVDF) membrane with wet electroblotting. The membrane was blocked with 5% bovine serum albumin (BSA) in a mixture of Tris-buffered saline and Tween-20 (TBST) for 1 h. Then it was incubated with β-actin (1:1000) and primary antibodies against TNFR1 (1:1000, Abcam, MA, USA), NF-κB p65 (1:2000, Abcam, MA, USA), ERK1/2 (1:1000, Abcam, MA, USA), p-ERK1/2 (1:1000, Abcam, MA, USA), IL-6 (1:1000, Abcam, MA, USA), Aβ (1:1000, Abcam, MA, USA) overnight at 4°C. Then the blots were incubated with the secondary antibody at room temperature for 1 h. When the immunodetection was finished, ECL assay kit (Nanjing, China) was used to visualize the protein bands which were then analyzed by Gel Image software version 4.00 (Tanon, China).

This was analyzed as in Ref. [36 ]. Transversal 400 μm thick cerebellar slices were added to tubes containing ice‐cold Krebs buffer with or without 2 mM BS3 (Pierce, Rockford, IL) and incubated for 30 min at 4°C. Cross‐linking was terminated by adding 100 mM glycine (10 min, 4°C). The slices were homogenized in lysis buffer (66 mM Tris–HCl pH 7.4. 1% SDS, 1 mM EGTA, 10% glycerol, 0.2 mg/ml leupeptin, 1 mM NaF, 1 mM Na‐orto‐vanadate) by sonication for 20 s. Samples treated with or without BS3 were analyzed by Western blot using antibodies against TNFR1 (Abcam, 1:1000), P2X4 (Invitrogen, 1:500), TrkB (Abcam, 1:500), KCC2 (Millipore, 1:1000) and GAT‐3 (Abcam, 1:1000). The surface expression of these proteins was calculated as the difference between the intensity of the bands without BS3 (total protein) and with BS3 (non‐membrane protein).³⁷

For immunohistological staining, tissue samples were first rehydrated in reverse order as described in Fixation and Processing of Tissue Samples. Subsequently, epitopes were exposed by boiling the samples in EDTA buffer (pH 8) for 45 min, followed by a permeabilization step in Triton X-100 in PBS +/+ for 5 min at room temperature. The tissue sections were framed using a PAP pen (Kisker Biotech GmbH & Co. KG, Steinfurt, Germany) to provide a hydrophobic barrier and were blocked afterwards for 30 min at room temperature in PBS containing 5% goat serum and 1% bovine serum albumin. Samples were then incubated with primary antibodies raised against claudin-1, -2, -4, occludin, ZO-1 (Thermo Fisher Scientific), TNFR-1 (abcam, Berlin, Germany), and TNFR-2 (antibodies-online GmbH, Aachen, Germany) for 1 h at 37°C, followed by four washing steps in blocking solution. Subsequently, secondary goat anti-rabbit Alexa Fluor-488, goat anti-mouse Alexa Fluor-594, and DAPI for the staining of nuclei were added to the samples, which were incubated again for 1 h at 37°C. Another four washing steps in blocking solution were carried out, followed by one wash in distilled water, and finally the sections were mounted in ProTaqs Mount Fluor (Biocyc, Luckenwalde, Germany). Microscopic analysis was carried out using a Zeiss 710 confocal microscope (Zeiss, Oberkochen, Germany).

After the ex vivo treatments, slices were homogenized by sonication for 20 s in buffer (Tris-HCl 66 mM pH 7.4, SDS 1%, EGTA 1 mM, glycerol 10%, leupeptin 0.2 mg/mL, NaF 1 mM, Na orto-vanadate 1 mM). Homogenates were subjected to electrophoresis and immunoblotting as described by Felipo et al. (24 (link)). Primary antibodies used were against BDNF (1:1000, Invitrogen), CCL2 (1:1000, Proteintech), Glutaminase 1 (1:1000, Novus Biologicals), GAT3 (1:1000, Synaptic Systems), HMGB1 (1:1000, Abcam), KCC2 (1:1000, Millipore), TNFα (1:500, RD systems), TNFR1 (1:500, Abcam) and TrkB (1:500, Abcam). Anti-actin (1:5000, Abcam) was used as protein loading control. Secondary antibodies were anti-rabbit, anti-rat, or anti-goat IgG conjugated with alkaline phosphatase (1:4000, Sigma). Membranes were scanned and band intensities were quantified using Alpha Imager 2200 version 3.1.3.