Protease and phosphatase inhibitor cocktail

RAW264.7 cells [treated with LPS alone (1 μg/ml) or together with 3α,5α-THP (1 μM) or pregnenolone (1 μM) for 24 hrs] were exposed to chemical protein crosslinking with membrane-permeable DSP (1 mM,Thermo Fisher Scientific, Cat. # PG82081) for 20 min on ice as previously described^{94 (link)}. The crosslinker was quenched in 1 M Tris buffer (pH 7.5), proteins were centrifuged at 21,000 × g for 15 min and extracted with Pierce IP Lysis Buffer (Thermo Fisher Scientific, Cat. # 87787) supplemented with protease and phosphatase inhibitor cocktails (Sigma). VTA micropunch proteins were extracted with CelLytic MT (Sigma Aldrich, St. Louis, MO, USA, Cat. # C3228) supplemented with protease and phosphatase inhibitor cocktails (Sigma) and co-immunoprecipitation was conducted as previously described^{43 (link),95 (link)}. Proteins were resolved by SDS-polyacrylamide gel electrophoresis, transferred to PVDF membranes and immunoblotted with MD-2, HMGB1, MYD88, GABA_AR-α2, TLR2, or TLR4 antibodies. The full-length gels showing specific TLR4/MD-2 protein interaction in RAW246.7 cells and TLR4/α2 and TLR4/MyD88 interaction the VTA are shown in SI, Figure S3.

M38 parental or Lingo2 KO cell lines (1 × 10⁵ per well) were seeded in 24-well plates and cultured for 48 h. in DMEM-10. Cells were then washed and cultured in serum-free medium for 24 h. Cells were treated with TFF3-Fc (1ug/ml) for 0, 5 min, 15 min, 30 min, and 1 h and were lysed with buffer containing [1% Triton X-100, 150 mM NaCl,10 mM Tris (pH 7.4), 1 mM EDTA, protease and phosphatase inhibitor cocktail (Sigma)]. Mouse colon tissues from WT or Lingo2 KO mice were lysed with RIPA buffer containing protease and phosphatase inhibitor cocktail (Sigma). 15ug samples were loaded and separated in 4–12% PAGE gel (Invitrogen). The following primary Abs were used for immunoblotting assays: mouse anti-Stat3 (Cell signaling,1:5000), rabbit anti-pStat3(Y705) (cell signaling, 1:1000), rabbit anti-EGFR (Millerpore,1:1000), rabbit anti-pEGFR (Y1068) (Abcam,1:1000); mouse anti-actin (Santa Cruz, 1:2500). Uncropped blots are shown in the Source Data File.

Frozen gastrocnemius was crushed using a mortar and a pestle cooled with liquid nitrogen. For the extraction of total protein, 50 mg of pulverized tissue was homogenized in a buffer containing 15 mM HEPES, 10% glycerol, 0.5% NP-40, 250 mM NaCl, 1 mM EDTA, 1 mM phenylmethanesulfonylfluoride (PMSF) and a phosphatase- and protease-inhibitor cocktail (Sigma-Aldrich, St. Louis, Mo, USA). Extracts were incubated 15 minutes on ice and centrifuged 15 minutes at 12 000 rpm and 4 °C. Supernatants were collected and protein concentration was determined by the BCA method.
For the preparation of nuclear extracts, 50 mg of pulverized frozen tissue was homogenized with a Dounce tissue grinder in a hypotonic buffer containing 20 mM HEPES, 5 nM NaF, 10 μM Na2MoO4, 0.1 mM EDTA, 0.01% NP-40, 1 M dithiotreitol (DTT) and a phosphatase- and protease-inhibitor cocktail (Sigma-Aldrich, St. Louis, Mo, USA). Extracts were centrifuged 10 minutes at 850 g and 4 °C. Cell pellets were gently re-suspended in hypotonic buffer, incubated 15 minutes on ice and centrifuged 30 seconds at 14 000 rpm and 4 °C. Nuclear pellets were then re-suspended in 50 μl of Complete Lysis Buffer (Active Motif, La Hulpe, Belgium), incubated 30 minutes on ice in a rocking platform and centrifuged 10 minutes at 14 000 rpm and 4 °C. Supernatants were collected and protein concentration was determined by the Bradford method.

Whole-cell lysates were collected by adding SDS-PAGE sample loading buffer. After sonication, the samples were boiled (10 min) and proteins were separated by SDS-PAGE and, then, they were transferred to nitrocellulose membranes and analyzed using immunoblotting (ECL Plus, GE Healthcare, Barcelona, Spain). For immunoprecipitation assays of cell extracts, the cells (~ 5 × 10⁶) were lysed in 500 μl of RIPA lysis buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% NP40, 1% sodium deoxycholate, 0.1% SDS, 5 mM EDTA) supplemented with protease and phosphatase inhibitor cocktails (Merck) and 50 µM IOX1 (a histone demethylase inhibitor; Merck). The cell extracts were cleared by centrifugation (20,000×g for 15 min). The extracts were precleared in 30-min incubations with 20 μl of Pure Proteome Protein G Magnetic Beads (Merck) at 4 °C while being rotated. The antibodies (as indicated in the figure legends) were then added to the precleared extracts. After incubation for 1 h at 4 °C, 50 μl of Pure Proteome Protein G Magnetic Beads were added, and the extracts were further incubated for 20 min at 4 °C with rotation. After extensive washing, the bound proteins were analyzed using western blots. The unbound extracts were used as the positive inputs to determine protein loading. Cytosolic extracts were obtained using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific).

The colon tissues were lysed with RIPA lysis buffer containing protease and phosphatase inhibitor cocktails (Merck, Darmstadt, Germany) and the proteins were detected by Western blot as previously described [28 (link)]. The antibodies used include rabbit anti-MANF (Abcam, #ab126321, 1:1000), rabbit anti-CHOP (Santa Cruz, #sc-793, 1:1000), rabbit anti-IL-12 (Bioss, #bs-0767R, 1:1000), rabbit anti-BATF2 (Thermo, #PA5-37138, 1:500), rabbit anti-cleaved caspase-3 (Affinity, #AF7022, 1:1000), and mouse anti-GAPDH (Elabscience, #E-AB-20032, 1:1000). The signal was detected by ECL solution (Beyotime, Shanghai, China, #P0018S), and the images were visualized on ChemiDoc (Bio-Rad, Hercules, CA, USA). The band densities were analyzed by ImageJ.