Lysis buffer

Adipose‐derived mesenchymal stem cells were isolated from inguinal subcutaneous adipose tissue as previously described.7 Briefly, adipose tissues were cut into small pieces in phosphate‐buffered saline (PBS) on ice. The minced tissue was then digested with 1 mg/mL Type 1 collagenase (Worthington‐Biochem, LS004196) at 37°C for 1 hour. The digested tissue was filtered through a 70 µm mesh (Corning, 431751) to remove tissue debris. The cell suspension was then centrifuged at 600 g for 15 minutes to remove collagenase. The cell pellet was plated in 100 mm dishes and incubated with 1× lysis buffer (Beyotime, C3702) at room temperature for 10 minutes to lyse red blood cells. After lysis of red blood cells, the pellets were maintained in mouse ADSCs complete medium (Cyagen, MUBMD‐90011) at 37°C in an atmosphere with 5% CO₂. Medium was changed after 24 hours and then every second day. ADSCs were used for subsequent experiments from the second passage. Recombinant mouse IL‐11 protein (R&D Systems, 418‐ML), Stattic (Selleck, S7024) and anti‐IL‐11Rα (R&D systems, AF490) were used to treat ADSCs.

Rat hippocampal neurons were cultured in 6-well plates at an initial concentration of 2 × 10⁶ cells per well. After culturing for 9–10 days, the neurons were treated with 10 μM manidipine, 5 μM KN-62, or 10 μM H89 for 10 min, or left untreated prior to treatment with 2 mM BHBA for another 1 or 12 h. Subsequently, the cells were lysed using 1× lysis buffer (Beyotime, China) containing protease inhibitor cocktail 2 (Roche, Switzerland) directly in the plates to obtain whole-cell lysates. Proteins were separated on SDS/PAGE gels and electroblotted onto PVDF membranes (GE Healthcare, United States). The membranes were blocked with 5% (w/v) BSA (bovine serum albumin; Sigma-Aldrich, United Kingdom) for 1 h at room temperature, and subsequently incubated with primary antibodies (tri-methyl histone H3 (Lys4) rabbit mAb, ubiquityl-histone H2A (Lys119) rabbit mAb, phospho-CaMKII (Thr286) rabbit mAb, phospho-CREB (SER133) rabbit mAb, WDR5(A-6), anti-BDNF antibody, anti-beta-actin) at 4°C overnight. Then, TBST was used to wash the membranes 3 times, followed by incubation with the horseradish peroxidase (HRP)-conjugated secondary antibody (Abcam, United Kingdom) for 1 h at room temperature. The protein bands were developed using the ImmobilonTM Western Chemiluminescent HRP Substrate (Millipore, United States) and photographed using the FlorChem FC2 System (Alpha Innotech, United States).

Protein extraction was carried out with a lysis buffer (Beyotime Institute of Biotechnology, Haimen, China). Proteins were quantified by an acid-based Protein Assay Kit (Thermo), followed by separation with 10% sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transfer to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). Afterward, 5% skim milk was used to block (2 h) the non-specific binding sites of membranes. Membranes were subsequently incubated with indicated primary antibodies overnight against PD-L1 (1:1000, ab213524, Abcam, Cambridge, UK), FOSL1 (1:1000, ab252421, Abcam), or PTBP1 (1:5000, ab134950, Abcam), followed by incubation with horseradish peroxidase-conjugated secondary antibody (1:5000, ab205718, Abcam). Membranes were washed with PBS three times, and signals were visualized by an ECL reagent (Millipore, Bedford, MA, USA). Protein expression was analyzed using ImageJ software 6.0 (National Institutes of Health, Bethesda, MD, USA). GAPDH (1:2000, ab8245, Abcam) and β-actin (1:5000, ab6276, Abcam) acted as internal controls.