Phenyl methyl sulfonyl fluoride (pmsf)

Each mice's hippocampal tissue was added radioimmunoprecipitation assay (RIPA) lysate buffer (Beyotime, Shanghai, P RChina) with 1 mM phenylmethanesulfonyl fluoride (PMSF, Beyotime) and 1 mM phosphatase inhibitor cocktail (PMSF, Beyotime), grinded with a tissue grinder and lysed on ice for 1 h. The lysates were collected and centrifuged at 12000 rpm for 15 min. Total proteins were quantified using a BCA protein assay kit (Beyotime). All samples adjusted to have the same concentration, mixed with a 5 × loading buffer and denatured at 95 °C.
Samples were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes. After blocking with 5% nonfat milk for 1.5 h and then incubated overnight at 4 °C with a primary antibody (including the following antibodies: Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb, Cell Signaling Technology, Cat. No. 12041S, 1:1000; Phospho-Glucocorticoid Receptor (Ser211) Antibody, Cell Signaling Technology, Cat. No. 4161S, 1:1000; BDNF Antibody, Cell Signaling Technology, Cat. No. 47808S, 1:1000; Anti-TrkB Rabbit pAb; Phospho-TrkB(Y817) Antibody; PI3 Kinase p85 alpha Antibody, Abmart, Cat. No. T40115F, 1:2000; Phospho-PI3-kinase p85-alpha/gamma (Tyr467/199) pAb, Abmart, Cat. No. T40065F, 1:2000; AKT1/2/3 Antibody, Abmart, Cat. No. T55561F, 1:2000; Phospho-Akt (Ser473) Antibody, Abmart, Cat. No. T40067F, 1:2000; mTOR (7C10) Rabbit mAb, Cell Signaling Technology, Cat. No. 2983S, 1:1000; Phospho-mTOR (Ser2448) (D9C2) XP® Rabbit mAb, Cell Signaling Technology, Cat. No. 5536S, 1:1000; Synaptophysin Antibody, Abmart, Cat. No. T55273S, 1:2000; PSD95-Specific, DLG4 Polyclonal antibody, Proteintech, Cat. No. 20665-1-AP, 1:6000; Anti -GAPDH Rabbit pAb, Servicebio, Cat. No. GB11002-100, 1:2000; Anti -beta Tubulin Rabbit pAb, Servicebio, Cat. No. GB11017-100,1:2000; Recombinant Anti - beta Actin antibody (Rabbit mAb), Servicebio, Cat. No. GB15003-100,1:2000). Blots were subsequently washed and incubated with secondary antibodies (HRP Conjugated AffiniPure Goat Anti-Rabbit IgG (H + L), Boster Bio, Cat. No. BA1054,1:5000) for 1.5 h. The blots were visualized using an ECL reagent (Merck Millipore) and the chemiluminescent signals were detected and analyzed using a ChemiDoc XRS+ (Bio-Rad, United States) image analysis system. The intensity of the protein bands was analyzed using Image Lab 6.0 software: Corresponding target proteins and internal reference proteins were framed and analyzed by the software to obtain gray scale values. The ratio of the target protein to the internal reference protein is the expression of the corresponding protein.

The cells were lysed in RIPA buffer (Beyotime, China, P0013B) supplemented with 1% PMSF (Beyotime, China, ST506) for 10 min. Protein concentrations were determined via a bicinchoninic acid assay (Beyotime, China, P0012S). The proteins were subsequently separated via 10% or 12.5% SDS‒PAGE (Epizyme, China, PG112, PG113) and subsequently transferred to polyvinylidene fluoride (PVDF) membranes (0.2 μm; Bio-Rad, USA). After blocking with 5% skim milk (Beyotime, China, P0216-300 g) for 120 min, the membranes were incubated overnight at 4 °C with the following primary antibodies: GPX4 (Affinity, USA, DF6701), ACSL4 (Affinity, USA, DF12141), LPCAT3 (Proteintech, China, 67882-1-Ig), P38 (Proteintech, China, 14064-1-AP), p-P38 (Proteintech, China, 28796-1-AP), JNK (Proteintech, China, 51151-1-AP), p-JNK (Proteintech, China, 80024-1-RR), GAPDH (Proteintech, China, 10494-1-AP) and β-actin (Wanleibio, China, WL01372). The membranes were subsequently incubated with secondary antibodies (Wanleibio, China, WLA023a) for 1 h. The blots were visualized via enhanced chemiluminescence reagents (Beyotime, China, P0018S). The density of each band was quantified via ImageJ version 1.51.

Cells or tissue proteins were lysed on ice for 20 min using RIPA lysis buffer (Beyotime, Beijing, China) containing 100 mM PMSF (Beyotime, Beijing, China). For the extraction of phosphorylated proteins, a phosphatase inhibitor (Epizyme, Shanghai, China) was added at a ratio of 100:1. Cellular or tissue debris was removed by centrifugation at 12,000 g for 15 min. Protein concentration was quantified using the bicinchoninic acid (BCA) method, and each sample was adjusted to a total protein content of 100 μg. Subsequently, 5× SDS-PAGE loading buffer (Beyotime, Beijing, China) was added to the samples, followed by denaturation at 95 °C for 10 min. Equal amounts of protein (25 μg) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene fluoride (PVDF) membranes (Thermo Fisher Scientific, Massachusetts, Waltham, USA). The membranes were blocked at room temperature for 1 h with 5% nonfat milk prepared in Tris-buffered saline containing 0.05% Tween-20 (TBST). The membranes were then incubated overnight at 4 °C with the following primary antibodies: α-SMA (1:2000, 42 kDa, Abcam, Cambridge, MA, USA), Fibronectin (1:2000, 285 kDa, Abcam, Cambridge, MA, USA), E-cadherin (1:20000, 130 kDa, Proteintech, Wuhan, China), β-actin (1:2000, 42 kDa, Abcam, Cambridge, MA, USA), Phosphorylated Spleen Tyrosine Kinase (p-Syk, 1:1000, 72 kDa, Cell Signaling Technology, Danvers, MA, USA), Inhibitor of Kappa B Alpha (IκBα, 1:1000, 36 kDa, Cell Signaling Technology, Danvers, MA, USA), Phosphorylated IκBα (p- IκBα, 1:1000, 36 kDa, Cell Signaling Technology, Danvers, MA, USA), or Alpha-tubulin (α-Tubulin, 1:1000, 55 kDa, Cell Signaling Technology, Danvers, MA, USA). After washing, the membranes were incubated with HRP-conjugated goat anti-rabbit IgG H&L (1:5000, Proteintech, Wuhan, China) or goat anti-mouse IgG H&L (1:5000, Proteintech, Wuhan, China). The antibody-antigen complexes were detected using the Amersham ECL detection system (Amersham, UK). Protein bands were quantified using ImageJ software (NIH, Bethesda, MD, USA), and the band intensities were normalized to β-actin or α-Tubulin.