Phenylmethylsulfonyl fluoride

HUVECs treated with 20μM DHA were collected at different time points. To generate a positive control for p38 MAPK activation, a group of HUVECs were treated with 1 μg/ml anisomycin for 1 h. Cell lysates were prepared in radioimmunoprecipitation assay buffer [20 mM Tris (pH 7.5), 150 mM NaCl, 50 mM NaF, 1% NP-40, 0.1% deoxycholate, 0.1% SDS and 1 mM EDTA] (Santa Cruz Biotechnology, Inc.), supplemented with 1 mM phenylmethylsulfonyl fluoride and 1 μg/ml leupeptin (Santa Cruz Biotechnology, Inc.). Cleared cell lysates were subjected to SDS-PAGE using 10% polyacrylamide gel and transferred to polyvinylidene fluoride membranes. Membranes were blocked with 2.5% non-fat milk, and incubated with primary antibodies at 4°C overnight in phosphate-buffered saline Tween-20 (Santa Cruz Biotechnology, Inc.). The primary antibodies used were total-p38, phospho-p38 (Cell Signaling Technology, Inc.) and β-actin (Sigma-Aldrich, St. Louis, MO, USA). Immunoreactivity was visualized with horseradish peroxidase-conjugated secondary antibodies and an enhanced chemiluminescence reagent (Santa Cruz Biotechnology, Inc.). The blots were analyzed using a Bio-Rad imaging system (Bio-Rad, Hercules, CA, USA).

Two million CD4 T-naïve cells from from young (aged 20–35 years) or older (aged 65–80 years) healthy donors or naïve CD4 T cells transfected with siYY1, siYY1/pre-miR-181a, siYY1/siDUSP6, siYY1/siSIRT1, or control siRNA and stimulated by anti-CD3/CD28 were lysed on ice with RIPA buffer (Thermo Scientific) supplemented with the phosphatase inhibitor sodium orthovanadate and the proteinase inhibitor phenylmethylsulfonyl fluoride (Santa Cruz Biotechnology). Ten micrograms total protein was separated on 4–15% gradient sodium dodecyl sulfate gels and transferred to polyvinylidene difluoride membranes. Membranes were incubated with primary antibodies specific for YY1, DUSP6, SIRT1, and pERK, respectively, then horse radish peroxidase-labeled secondary antibody and developed with Pierce ECL Western blotting substrate (Thermo Fisher Scientific). For loading control, the membrane was stripped with stripping buffer (Invitrogen) and re-probed with anti-β-actin antibodies. Uncropped immunoblot images are included in Supplementary Figs. 2–4.

Western blotting was performed in accordance with the manufacturer's specifications. After OGD treatment, C8-D1A and BV2 cells were collected. Cells were lysed in RIPA buffer containing phenylmethylsulfonyl fluoride (Santa Cruz, CA, USA) for protein extraction, followed by centrifugation at 12,000 rpm for 15 min at 4 °C to remove insolubles. Protein concentration was quantified using the BCA Protein Assay Kit (Beyotime technology, China). Equal amounts of protein were loaded onto SDS-PAGE gels for electrophoresis, followed by transfer to polyvinylidene fluoride membranes. After blocking with 5% non-fat milk for 2 h, the membranes were incubated overnight at 4 °C with Nrf2, IL-1β (1:2000, 16,806–1-AP, Protrintech) and HO-1 (1:1000, 10,701–1-AP, Protrintech) antibody. After primary antibody incubation, membranes were washed at least three times with TBS containing 0.2% Tween-20, then incubated with the appropriate secondary antibody at room temperature for 1.5 h. The membranes were then washed three times with TBST. Finally, detection of protein bands was conducted using an enhanced chemiluminescence reagent and imaged with an imaging densitometer. Data analysis was performed using ImageJ software.

CD4⁺CD45RA⁺ cells were plated at 2 × 10⁶ cells per condition for Western blotting. The cells were lysed with lysis buffer containing 2.5 mM sodium pyrophosphate, 1 mM NA3VO4, and 1 mM phenylmethylsulfonyl fluoride (Santa Cruz Biotechnology). The cell lysates were resolved with 5–15% gradient SDS-PAGE (Bio-Rad) and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% milk in TBS (20 mM Tris and 500 mM NaCl) and 0.1% Tween 20 at room temperature for 1 h followed by overnight incubation at 4°C with primary Abs against IL-1RI (Abcam), IRF4 (Santa Cruz Biotechnology), RORc (Abcam), β-actin (Sigma-Aldrich). Secondary HRP-conjugated Ab (Santa Cruz Biotechnology) was added at a dilution of 1/2000 for 1 h and the protein bands were detected with an ECL Detection System (Santa Cruz Biotechnology).

Cholesterol (Chol), sphingomyelin (SM), and phospholipids (PLs): 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylCholine (POPC), 1-palmditoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylserine (POPS), were obtained dissolved in chloroform from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). Cholesterol analog cholestane spin label (CSL), HEPES, Tris-HCl, NaN₃, sodium chloride (NaCl) lysozyme, deoxycholic acid, and DNase I were obtained from Sigma Aldrich (St. Louis, MO, USA). Isopropyl-1-thio-β-D-galactopyranoside (IPTG), ampicillin, phenylmethylsulfonyl fluoride, and polyethyleneimine were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Recombinant human αAc and αBc were expressed and purified, and the reconstituted 3:1 heteromeric complex of αAc to αBc (i.e., αABc) was prepared using the methods described in Section 4.2 and stored in HEPES buffer (10 mM HEPES, 100 mM NaCl, pH = 7.4). All preparations of α-crystallin (αAc, αBc, and αABc) and the Chol/MHLL membranes, as well as associated binding studies, were performed in HEPES buffer (10 mM HEPES, 100 mM NaCl, pH = 7.4).

ucMGP concentrations in GGCX dermal fibroblasts were quantified using a human ucMGP ELISA Kit according to the manufacturer’s protocol (#CSB-EC013789HU, Cusabio Biotech Co., Wuhan, China). Briefly, cells (6.4 × 10⁵) isolated from three controls and one GGCX patient were separately seeded on 6-well plates. When they reached early confluency, cells were washed with cold PBS twice and lysed on ice for 5 minutes with RIPA buffer (#9806S, Cell Signaling Technology) containing protease inhibitors (Protease Inhibitor Cocktail Set I; Calbiochem) and 1 mM phenylmethylsulfonyl fluoride (Santa Cruz). The lysate were collected using a cell scraper and vortexed for 30 seconds. After centrifugation at 12,500 rpm for 10 minutes at 4°C, protein concentrations in the supernatants were quantitated using a Quick Start Bradford protein assay (Quick Start BSA standard set, Bio-Rad Laboratories, Inc.). Samples (approximately 40 μg of protein) were mixed with the sample diluent, and the ucMGP content was assessed. Reactions were stopped by addition of acidic stop solution, followed by measurement of OD at 450 nm in triplicate (Labsystems Multiskan MS #352, Thermo Fisher Scientific Inc.). The values were adjusted by the protein contents, which were determined from the same plates.