M iggκ bp hrp

Immunohistochemical analyses were performed as previously described³⁰ , with some modifications. Before immunostaining, antigen retrieval was performed via incubation of specimens in 0.1% pepsin (Nacalai Tesque) in 0.5 M acetic acid (Sigma-Aldrich) at 37 °C for 1 h in a humid chamber. Deparaffinized sections were rehydrated and incubated in 0.3% hydrogen peroxide (H₂O₂; Wako Pure Chemical) in PBS to block endogenous peroxidase activity. Then, non-specific binding sites were blocked using 2% BSA (Wako Pure Chemical), 0.1% Tween20 (Sigma-Aldrich), and 0.01% Triton-X (Wako Pure Chemical) prior to incubation in the primary antibody at 4 °C overnight. Then, the samples were incubated in mouse IgGκ light chain binding protein conjugated to horseradish peroxidase (m-IgGκ BP-HRP: Santa Cruz Biotechnology, CA, USA) as the secondary antibody for 60 min. Next, the samples were washed with PBS and incubated in DAB substrate working solution (Roche Applied Science, Mannheim, Germany) for 5–15 min. Subsequently, hematoxylin was used to counterstain nuclei for visualization. The primary antibodies used in this study were an anti-type II collagen monoclonal antibody (M2139: Santa Cruz Biotechnology) and anti-type X collagen monoclonal antibody (X53: Invitrogen/Thermo Fisher Scientific).

To determine the protein levels of SOCS3, Claudin-5, and phosphorylated STAT3, total proteins of the brain tissue were extracted using the M-PER Mammalian Protein Extraction Kit (Pierce Biotechnology, Inc., Waltham, MA), and their concentrations were quantified using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). The proteins were separated on 10% SDS polyacrylamide gels for 110 min at 80 V at RT and then transferred to a PVDF membrane for 90 min at 80 V at 4 °C. After blocking with 3% BSA/0.1% TBS-T at RT, the membrane was incubated with each of anti-SOCS3 (Abcam, Cambridge, UK), anti-Claudin-5 (Invitrogen, Carlsbad, CA), anti-STAT3 (Santa Cruz Biotechnology, Dallas, TX), anti-pSTAT3 (Abcam, Cambridge, UK), and anti-β-actin (Santa Cruz Biotechnology) at 4 °C overnight. The m-IgGκ BP-HRP (Santa Cruz Biotechnology) or HRP–goat anti-rabbit IgG (Thermo Fisher Scientific) antibody was used as a secondary antibody. After washing, the membrane was incubated with Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific) for 30 s. The signal was detected with an Amersham Imager 600 (GE Healthcare, Pittsburgh, PA), and the signal intensity was evaluated using ImageJ software.

Approximately 10⁶ cells were harvested, by centrifugation at 300×g for 5 min, and processed for total protein isolation and western blot analysis. Pelleted cells were washed twice with PBS and lysed in buffer A (10 mM Tris-Cl pH 8.0, 150 mM NaCl, 2% SDS). The lysate appeared highly viscous due to DNA release and, therefore, it was sonicated for 30 s. The samples were then processed for protein quantitation using the BCA assay kit. One volume of protein sample was, consequently, mixed with equal volume of laemmli sample buffer (4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.004% bromophenol blue and 0.125 M Tris HCl, pH 6.8) and boiled for 5 min at 95 °C. Twenty µg of total protein material was then loaded on a denaturing 12% SDS-PAGE gel and run until separation. Subsequently, proteins were transferred to a PVDF membrane and blotted with primary antibodies overnight at 4 °C in a shaker and with secondary antibody for 1 h at room temperature. The antibodies used were from Santa Cruz Biotechnology (Finnell Street Dallas, Texas 75220, USA): Hemoglobin β/γ/δ/ε (sc-390668), β-actin (sc-47778), α- tubulin (sc-51503) and m-IgGκBP-HRP (sc-516102).

Protein extraction (SDS–PAGE, semi-dry blotting and immunodetections were carried out as described previously (Hammel et al., 2020 (link)). Primary antibodies used for immunodetection were mouse anti-HA (Sigma H9658, 1:10,000) and rabbit anti-GFP (Roche, Cat. No. 11814460001, 1:5,000). The secondary antibody was m-IgGκBP-HRP (Santa Cruz Biotech sc-516102, 1:10,000). Densitometric band quantifications after immunodetections were done with the FUSIONCapt Advance program (PEQLAB).

Polystyrene 96-well plates were sensitized (Maxisorp Nunc) with 50 µl of protein (10 µg), serum (1:2 dilution), or VEGF (Santa Cruz Biotechnology) standard (0.001 ng-1ng) in bicarbonate buffer (in duplicate) and incubated at 4°C overnight following manufacturer instructions. Subsequently, the plates were washed three times with a wash solution. 200 µl of the blocking solution (1%) was added and incubated for one hour at 4°C. Next, 50 µl of anti-VEGF C-1 antibody (sc-7269, Santa Cruz Biotechnology) (1:200) was incubated for one hour at 4°C. For recognition of anti-VEGF IgG, 50 µl of m-IgGκ BP-HRP (sc-516102 Santa Cruz Biotechnology) was added (1:400) for 2 h at room temperature. Finally, after washing, the enzyme-substrate reaction was performed with 50 µl of the chromogen solution. The reaction was stopped after 20 min with 50 µl of 2 N sulfuric acid. The absorbances were determined at 492 nm in the Stat Fax 4200 microplate reader (Awareness Technology). All cytokine and VEGF concentrations were calculated with interpolation from a standard curve. The determination of TES concentration, dilutions of sera, and antibodies was developed after the standardization of the respective ELISAs.

Whole-cell protein was extracted by RIPA Lysis Buffer with Halt™ Protease and Phosphatase Inhibitor Single-Use Cocktail, EDTA-Free (100×), and protein concentration was measured by Pierce BCA Protein Assay Kit (All from Thermo Scientific™, Waltham, MA, USA). Total protein was separated by SDS-PAGE, transferred to PVDF membrane (Millipore, Billerica, MA, USA), and detected by SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Scientific™, Waltham, MA, USA). Primary and secondary antibodies used in the experiments were listed as follows: anti-PERK, anti-Phospho-PERK (Thr980), anti-glucose-regulated Protein 78 (GRP78), anti-CHOP, anti- eukaryotic initiation factor 2 alpha (eif2α), anti-elF2 alpha (phospho S52), anti-cleaved-caspase-3, anti- B cell lymphoma 2 (BCL2)-associated X protein (BAX), anti-BCL2 (1:1000 dilution, Cell Signaling Technology, Danfoss, MA, USA), mouse anti-rabbit IgG-HRP, and m-IgGκ BP-HRP (1:3000 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA).