Af1015

Proteins were extracted by mixing RIPA lysate (R0010, Solarbio) and PMSF (P0100, Solarbio). The concentrations of proteins were assayed by the BCA kit (PC0020, Solarbio). According to different molecular weights, 5% concentrated gel and 8% separation gel concentrations were used in the SDS-PAGE. After transferred to a PVDF membrane, the proteins were blocked by prepared 5% (M/V) BSA (Biosharp, BS043, China) in TBST buffer for 1 h and incubated overnight at 4 °C with the following primary antibodies: α-SMA (1: 500), COL-I (1: 500, AF0134, Affinity), TGF-β1 (1: 1000, AF1027, Affinity), proliferating cell nuclear antigen (PCNA) (1: 500, A12427, Abclonal, China), p-MEK1/2 (1: 500, AP0209, Abclonal), MEK1/2 (1: 500, A4868, Abclonal), p-ERK1/2 (1: 500, AF1015, Affinity), ERK1/2 (1: 500, AF6240, Affinity) and GAPDH (1: 10000, 60004-1-Ig, Proteintech, China). Next, the membrane was incubated with goat anti-mouse IgG (1: 3000, SE131, Solarbio) or goat anti-rabbit IgG (1: 3000, SE134, Solarbio) secondary antibody for 40 min at 37 °C. At last, the specific protein bands were visualized with Western ECL Substrate (D1010, Solarbio).

Total protein was extracted from HMy2.CIR and DLBCL cells using ice-cold RIPA lysis buffer (Solarbio, Beijing, China) supplemented with proteinase inhibitors (Beyotime, China). Next, protein concentration was assessed using the BCA protein assay kit (Beyotime, China). Equal amounts (25 μg) of protein extracts were separated with 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Burlington, MA, USA). The PVDF membranes were blocked with 5% nonfat milk for 2 h at room temperature, following incubation with the primary antibodies against HK2 (1:1,000, BF0283, Affinity), ERK1/2 (1:2,500, AF0155, Affinity), phospho-ERK1/2 (Thr202/Tyr204) (1:1,000, AF1015, Affinity), Bax (1:2,000, ab182733, Abcam), Bcl-2 (1:1,000, AF6139, Affinity), caspase-3 (1:500, ab13847, Abcam), E-cadherin (1:2,000, 20874-1-AP, ProteinTech), N-cadherin (1:1,000, AF6139, Affinity), and GAPDH (1:5,000, AF7021, Affinity) at 4°C overnight. After washing three times with TBST, the membranes were incubated with an anti-rabbit or anti-mouse HRP-conjugated secondary antibody for 1 h. Finally, bands were detected using an enhanced chemiluminescence (Beyotime, China) system and analyzed using ImageJ (v1.8.0, National Institutes of Health, USA).

Total proteins from cultured hGFs were extracted using radioimmunoprecipitation assay (RIPA) lysis buffer on the ice. The protein concentration was determined via a Nanodrop. Proteins at 10 μL per lane were separated by 4–20% SDS-PAGE (Beyotime, China) and then transferred onto PVDF membranes (Merck, Germany), which were blocked with 5% bovine albumin and then incubated with primary antibodies to MAT2A (1:1000; 55309–1-AP; Proteintech, China), IL-1β (1:1000; BJ11258993; Bioss, China), TNF-α (1:400; GB11188; Servicebio, China), IL-6 (1:1000; 21865–1-AP; Proteintech, China), MCP-1 (1:1500; ab25124; Abcam, USA), P65 (1:1000; 8242S; CST, Germany), Erk (1:1500; GB11560; Servicebio, China), P38 (1:1000;8690S; CST, Germany), JNK (1:1000; AF6318; Affinity, USA), p-P65 (1:1000; 3033S; CST, Germany), p-Erk (1:1000; AF1015; Affinity, USA), p-P38 (1:1000; 4511S; CST, Germany), p-JNK (1:1000;4668S; CST, Germany) and β-ACTIN (1:50000; 66009-l-Ig; Proteintech, China). After the incubation step, membranes were washed again 3 times and incubated with secondary antibodies. Images were provided with ImageQuant LAS 4000 (USA). The intact optical density for the protein band was calculated by the ImageJ software.

Total protein was prepared by extracting the supernatant after lysing tissues or cells with RIPA lysis buffer (Elabscience, E‐BC‐R327) and subsequently adding a loading buffer (wshtbio, ES003). The proteins were resolved using a 12.5% SDS‐PAGE gel (Epizyme, Shanghai, China) and subsequently blotted onto 0.22 μm nitrocellulose membranes (Millipore, America). After incubation with 5% BSA for 1 hour at room temperature, the membrane was incubated with primary antibody overnight at 4°C. The primary antibodies used were as follows: anti‐GAPDH (1:1000; Elabscience, E‐AB‐40337), anti‐CCR6 (1:1000; SinoBiogical, #106771‐T32), anti‐IL‐17A (1:1000; Affinity, DF6127), anti‐CCL20 (1:1000; Affinity, DF2238), anti‐P65 (1:1000, Affinity, AF5006), anti‐Phospho‐P65 (1:1000, Affinity, AF2006), anti‐ERK1/2 (AF0155), anti‐Phospho‐ERK1/2 (1:1000, Affinity, AF1015), anti‐P38 (1:1000, Affinity, AF6456), anti‐Phospho‐P38 (1:1000, Affinity, AF4001), anti‐mTOR (1:1000, CST, 2983 T), anti‐Phospho‐mTOR (1:1000, CST, 5536 T), anti‐AKT (1:1000, CST, 4691 T) and anti‐Phospho‐AKT (1:1000, CST, 4060 T). Then the bands were incubated with the secondary antibodies (1:5000; Elabscience, E‐AB‐1003), which was derived from rabbit and conjugated with HRP. Subsequently, blots were captured with an ECL luminescence system (ClinX, Shanghai, China). The gels were quantified and analysed using ImageJ.

The mice were administered 1% pentobarbital sodium and decapitated. Then, bilateral hippocampal tissues were immediately collected. SH-SY5Y cells were collected after exposure. Cells and lysed tissues in RIPA buffer supplemented with protease and phosphatase inhibitors were collected. Protein concentrations were measured with BCA reagent (P0006, Beyotime Biotechnology, China). Protein samples (20 μg) were separated by SDS‒PAGE (10% separation gel) and transferred to a PVDF membrane (Merck and Co., Inc., Whitehouse Station, NJ, United States, Germany). After blocking with TBST containing 5% nonfat milk for 1 h, the membranes were incubated with Gpx4 (ab125066, 1:1000; Abcam), Hmox-1 (ab189491, 1:2000; Abcam), P2rx7 (ab259942, 1:1000, Abcam), phospho-Erk1/2 (AF1015, 1:1000; Affinity Biosciences), Erk1/2 (AF0155, 1:1000; Affinity Biosciences), phospho-p38 (AF4001, 1:1000; Affinity Biosciences), p38 (AF6456, 1:1000; Affinity Biosciences), phospho-Jnk (AF3318, 1:1000; Affinity Biosciences), and Jnk (AF6318, 1:1000; Affinity Biosciences) overnight at 4 °C. The next day, following three washes in TBST, the membranes were incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (A0208, 1:2000, Beyotime) or anti-mouse IgG (A0216, 1:2000, Beyotime) at room temperature for 1 h. The protein levels were normalized to GAPDH (#5174, 1:1000, Cell Signaling Technology).