Protease inhibitor cocktail

Trifluoroacetic acid (TFA), methanol, acetonitrile (ACN), formic acid (FA), NH₄HCO₃, urea, dithiothreitol (DTT), iodoacetamide (IAA), RIPA buffer, sodium pyruvate, L-glutamine, G418, lipopolysaccharide and non-essential amino acid solution were obtained from Sigma-Aldrich (St. Louis, MO, United States). Trypsin (V5280) was purchased from Promega (Madison, WI, United States). Ultrapure water was prepared by Milli-Q water purification system (Millipore, Bedford, MA, United States). The TMT6plex^TM Mass Tag Isobaric Labeling kit was purchased from Thermo Scientific^TM (Madison, WI, United States). Protease inhibitor cocktail was purchased from (GE Healthcare, Little Chalfont, United Kingdom).

The hippocampus was separated from the brain soon after the mice were sacrificed, and it was stored at −80°C for subsequent analysis. For protein extraction, the hippocampus was homogenized in lysis buffer containing protease inhibitor cocktail (10 μl/ml, GE Healthcare Biosciences, PA, United States). Then, the complex was centrifuged at 12,000 rpm, and the supernatant was obtained for the protein assay. The extracted protein was quantified with BCA reagents (Beyotime). For IP, 300 mg of protein from hippocampus was incubated with 1 mg of rabbit anti-ASK1 antibody at 4°C overnight. After that, the protein was further incubated with protein A magnetic bead slurry. Then, the protein was separated on a 10% or 12% gel, and transferred onto a PVDF membrane (Sigma-Aldrich, St Louis, MO). The membrane was blocked with 5% milk in TBS for 0.5 h and incubated with primary antibodies in TBS overnight at 4°C. After washing three times with TBST (TBS with 0.05% tween 20), the membrane was treated with horseradish peroxidase-conjugated secondary antibodies (1:3000) for 4 h at room temperature. Signals were visualized by ChemiDocXRS + Imaging System (BioRad). All experiments were repeated in triplicate using independently prepared tissue. The densitometric values of bands on Western blotting were obtained by Image J software and subjected to statistical analysis.

Protein extraction
was carried out from P. gingivalis cells (control
and treated) as previously reported.^{19 (link)} Briefly,
the cells were centrifuged for 5 min at 5,000g at
4 °C. Then, the supernatants were discarded, and the resulting
pellets were washed with phosphate-buffered saline (PBS). Subsequently,
the protein pellets were suspended in lysis buffer (0.5 mL; pH 8.8;
30 mM Tris buffer containing 7 M urea, 2 M thiourea, 2% Chaps, and
the protease inhibitor cocktail; GE Healthcare, Chicago, IL, USA)
on ice for 20 min. After that, samples were vortexed and sonicated
(3–4 times) for 1 min and recentrifuged at 10,000g at 4 °C for 3 min to remove cell debris. Finally, protein concentrations
were determined using the 2D-Quant Kit according to the manufacturer’s
instructions (GE Healthcare).

Sample preparation for proteomic analysis was performed as follows: the samples were lysed in a lysis buffer containing 1% sodium deoxycholate (SDC, Sigma), 100 mM Tris-HCl (pH 8.5) with a protease inhibitor cocktail (GE Healthcare) through ultrasonication with a Branson 1510 sonicator at 4°C for 1 min, duty cycle 10%. Protein concentration was estimated using the BCA assay (Sigma). Aliquots containing 300 μg of the protein material were diluted to 1 μg/μL with lysis buffer and Tris (2-Carboxyethyl) phosphine hydrochloride (TCEP, Sigma) and chloroacetamide (CAA, Sigma) were added to final concentrations of 10 and 30 mM, respectively. Cys-reduction and alkylation were achieved by heating the sample for 10 min at 85°C. Trypsin (Promega, United States) was added at a ratio of 1:100 w/w to the protein amount and incubated at 37°C for overnight. Then, the second Trypsin portion 1:100 w/w was added and the sample was incubated for 4 h at 37°C. Proteolysis was stopped by the addition of 1% trifluoroacetic acid. The precipitated SDC was removed using ethyl acetate (Masuda et al., 2008 (link)). The samples were purified using OASIS columns (Water) and analyzed using liquid chromatography-mass spectrometry (LC-MS).

DRGs (L4-L6) from a pool of six rats eight- to ten-weeks-old subjected to either SCI (dorsal hemisection) or conditioning lesion (CL) were sacrificed one week after SCI. DRG were homogenized in lysis buffer (20 mM 4-morpholinepropanesulfonic acid (MOPS), 2 mM ethylene glycol tetraacetic acid (EGTA), 5 mM ethylenediaminetetraacetic acid (EDTA), 30 mM NaF, 60 mM β-glycerophosphate, 20 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1% Triton X-100, 1% dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor cocktail (GE Healthcare, Carnaxide, Portugal)). Protein extracts (500 μg) were analyzed using the Kinexus phospho-site broad signaling pathway screen version 1.3 (KPSS-1.3, Kinexus Bioinformatics Corp, Vancouver, Canada). This screen examines 38 phosphorylation sites in 32 proteins with antibodies that recognize specific phosphorylated epitopes. The intensities of signals for target protein bands on the Kinetworks immunoblots were quantified as described [54 (link)]. Proteins with a fold change CL/SCI lower than 0.75 or higher than 1.25 were selected.

The levels of Aβ_1-42 in isolated microglia were determined using a commercial ELISA kit (Wako). Protein was extracted from isolated microglia in ice-cold 70% (v/v) formic acid (Wako, Tokyo, Japan) followed by centrifugation at 20,000 ×g for 1 h at 4°C. The supernatant was neutralized with a 20-fold dilution in 1M Tris buffer, and used for Aβ_1-42 ELISA. The concentration of Aβ_1-42 was normalized against the number of isolated microglia.
The levels of IL-6 and TNF-α in plasma were determined using a commercial ELISA kit (BioLegend, San Diego, CA, USA). The levels of CSF1 in plasma and brain were determined using a commercial ELISA kit (R&D systems, Minneapolis, MN, USA). The snap-frozen brain tissues were homogenized in ice-cold PBS containing 1% protease inhibitor cocktail (GE Healthcare UK Ltd., Buckinghamshire, UK) and 1% phosphatase inhibitor cocktail (Nacalai), followed by sonication for 5 min and centrifugation at 12,000 ×g for 10 min at 4°C. The supernatant was used for CSF1 ELISA. The total protein concentration in each sample was determined by a BCA assay kit (Thermo Fisher Scientific) and the concentration of target protein in tissues were reported as pictograms of cytokine relative to the protein content. Absorbance was measured using the iMark microplate reader (BIO RAD, Hercules, CA, USA), and data was analyzed using the Microplate Manager 6 software (BIO RAD).