Af4001

ARPE‐19 cells were lysed in a lysis buffer to extract total protein, and the protein concentration was measured by a bicinchoninic acid assay kit (Beyotime, Shanghai, China). Samples were loaded and separated in 10% Bis‐Tris‐polyacrylamide electrophoresis gels. Then, the proteins were transferred to PVDF membranes, which were blocked with 5% nonfat milk followed by incubation with primary antibodies at 4 °C overnight. After incubation with the secondary antibody for 1 h, the Western blot signals were detected by ECL Plus reagents. ImageJ was used to analyze the protein bands. Primary antibodies against Occludin (1:800, Cat No. 17590‐1‐AP), TLR4 (1:800, Cat No. 66350‐1‐Ig), p65 (1:500, Cat No. 10745‐1‐AP), JNK (1:500, Cat No. 24164‐1‐AP), p‐JNK (1:500, Cat No. 80024‐1‐RR) and MYD88 (1:800, Cat No: 23230‐1‐AP) were purchased from Proteintech. Antibodies against p‐p65 (1:1000, AF2006), GADD45G (1:1000, A10286), p‐p38 (1:1000, AF4001), p38 (1:1000, AF6456), GAPDH (1:3000, AF7021), β‐actin (1:3000, AF7018) and OR11H1 (1:1000, DF5186) were purchased from Affinity.

For western blot analysis, proteins were extracted from femoral tissues using RIPA Buffer (20101ES60, Yeasen, China). We used the BCA kit (BI-WB005, SBJBIO, China) for protein quantification. After electrophoresis, the protein was loaded onto PVDF membranes (PW0034, Leagene, China). Next, 5% bovine serum albumin (BL-082, SBJBIO, China) was added to seal the membranes (37 °C, 60 min). The membranes were then immersed in primary antibodies (4 °C, overnight) and subsequently in anti-rabbit secondary antibodies (31,466, Invitrogen, USA) or anti-mouse secondary antibodies (S0002, Affinity, USA) at 37 °C for 60 min. Protein visualization was performed using an ECL reagent (GK10008, GlpBio, USA) on an eZwest Lite Auto Imaging System (Genscript, USA). The primary antibodies of Nrf2 (1:2000, AF0639), heme oxygenase 1 (HO-1) (1:2000, AF5393), phospho-IKB alpha (Ser32/Ser36, 1:2000, AF2002), IKB alpha (1:2000, AF5002), phospho-NF-kB p65 (Ser536, 1:2000, AF2006), NF-kB p65 (1:2000, AF5006), phospho-extracellular regulated protein kinases ½ (ERK1/2) (Tyr204, 1:2000, AF1014), ERK1/2 (1:2000, AF0155), phospho-p38 mitogen-activated protein kinase (MAPK) (Thr180/Tyr182, 1:500, AF4001), p38 MAPK (1:1000, AF6456), and GAPDH (1:20,000, AF7021) were obtained from Affinity (USA). GAPDH was used as the loading control.

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).

Western blot assay was performed as described by Sale et al [19 (link)]. Total proteins were extracted from cells using RIPA lysate (R0010, Solarbio) containing phenylmethylsulfonyl fluoride protease inhibitor (PMSF, P0100, Solarbio). Then, protein concentration was detected with a BCA kit (PC0020, Solarbio). After separated by 5-10% SDS-PAGE, transferred to the PVDF membrane, blocked by nonfat milk (A600669, Sangon Biotech, China), proteins on the membrane were incubated overnight at 4°C with the primary antibodies: iNOS (1: 1000, A0312, Abclonal, China), COX-2 (1: 2000, A1253, Abclonal), MMP-1 (1: 2000, A1191, Abclonal and 1: 1000, DF6325, Affinity), MMP-3 (1: 2000, A11418, Abclonal), MMP-13 (1: 2000, A11148, Abclonal and 1: 1000, AF5355, Affinity), p-ERK1/2 (1: 1000, AF1015, Affinity, China), ERK1/2 (1: 1000, AF0155, Affinity), p-p38 (1: 500, AF4001, Affinity), p38 (1: 500, AF6456, Affinity), p-JNK (1: 1000, AF3318, Affinity), JNK (1: 500, AF6318, Affinity), p-p65 (1: 1000, AF2006, Affinity), p65 (1: 1000, AF5006, Affinity), and GAPDH (1: 10,000, 60,004-1-Ig, Proteintech, China). Next, the membrane was incubated with appropriate secondary antibody. Finally, membranes were detected by Western electrogenerated chemiluminescence (ECL) Substrate (D1010, Solarbio) and the images were analyzed by Gel-pro analyzer software (Media Cybernetics, CA, USA).