Cw0096

Protein extracts were prepared from SH-SY5Y cells or brain tissues by homogenization in RIPA buffer with protease inhibitors and then were centrifugated to eliminate particulates at 14,000 g for 10 min at 4°C. Protein concentration was measured by the BCA method. Samples were separated on SDS-PAGE and transferred to nitrocellulose membranes. Immunoblotting was carried out with the primary antibodies specific for NLRP3 (ab4207, Abcam), caspase-1 (ab1872, Abcam), IL-1β (16806-1-AP, Proteintech), HDAC6 (07-732, Millipore), tyrosine hydroxylase (TH; MAB318, Millipore), acetylated lysine (9441, Cell signaling), peroxiredoxin 2 (Prx2; ab109367, Abcam), and 4-Hydroxynonenal (4-HNE; ab46545, Abcam). The primary antibody against β-actin (CW0096, CWBIO) was used to correct the sample loading. Densitometry analysis was performed using AlphaEaseFC software.

Dissected hippocampal tissues were homogenized in RIPA buffer (1% NP‐40, 0.1% SDS, 0.5% PMSF, and 4% Roche protease inhibitor) by sonication. After centrifugating at 12,000g for 20 min at 4°C, the supernatant was collected and protein concentration was measured. A total cell extract containing ~30 μg of protein was subjected to SDS‐PAGE and then transferred onto NC membrane. The membrane was blocked by incubation with 5% skim milk for 90 min and then hybridized overnight with primary antibodies at 4°C. The primary antibodies used include anti‐ferritin light chain (ab109373, Abcam), anti‐ferritin heavy chain (ab183781, Abcam), anti‐TfR1 (13–6800, Invitrogen), anti‐Bcl2 (12789‐1‐AP, Proteintech), anti‐Bax (50599‐2‐Ig, Proteintech), anti‐brain‐derived neurotrophic factor (BDNF) (ab108319, Abcam), anti‐furin (ab183495, Abcam), and anti‐β‐actin (CW0096, CWBIO). After washing with tris‐buffer saline with 0.05% Tween‐20, the membrane was incubated at room temperature for 90 min with anti‐rabbit or anti‐mouse secondary antibody conjugated with horseradish peroxide (Zhongshan Biotechnology). Immunoreactive bands were detected by the enhanced chemiluminescence detection kit (Amersham, UK) and visualized by a Bio‐Rad chemiluminescence imager.

According to previous reports, proteins were detected in retinal tissue. In brief, whole retina tissues were lysed in RIPA buffer (Beyotime, P0013B) and mixed well. PMSF (Beyotime, ST505) was added before use so that the final concentration was 1 mM. After cell disruption, lysates were centrifuged at 10,000-14,000 g for 10 min. From the supernatant, protein concentration was determined using a Protein Concentration Detection Kit (Beyotime, P0006C). A total of 50 μg protein was loaded onto 6–10% SDS-PAGE gels (120 V electrophoresis for 100 min). Proteins were then transferred from gels to 0.45 μm PVDF membranes (Millipore, IPVH00010) under a constant flow of 250 mA for 2 h. PVDF membranes were sealed with 5% BSA and incubated with primary antibodies (CD31, Abcam, ab222783, 1:200; Brn-3a, Santa Cruz, SC8429, 1:100; β-Actin, CWBIO, CW0096, 1:1,000) at 4°C overnight. Then they were washed with TBST three times, and corresponding secondary antibodies were added and incubated at 37°C for 1 h, washed with TBST three times, and exposed by an ECL Chemiluminescence Kit (Thermo, 32209).

Total proteins were extracted with RIPA lysis buffer (Servicebio Technology, Wuhan, China). Thereafter, proteins were separated via SDS–polyacrylamide gel electrophoresis and then were subsequently transferred to polyvinylidene fluoride membranes. Subsequently, the membranes were incubated with primary antibodies for the following proteins: Akt (1 : 1000; 9272S, CST), p-Akt (Ser473; 1 : 1000; 4060S, CST), Rps6kb1 (1 : 1000; 9202S, CST), p-Rps6kb1 (Thr-389; 1 : 1000; 9205S, CST), Eif4ebp1 (1 : 1000; 9452S, CST), p-Eif4ebp1 (Ser-65; 1 : 1000; 9451S, CST), Yap (1 : 1000; 14074S, CST), p-Yap (Ser-127; 1 : 1000; 13008S, CST), Gapdh (1 : 2000; CW0100, CWBiotech, Beijing, China), and β-Actin (1 : 2000; CW0096, CWBiotech, Beijing, China). The membranes were then incubated with the corresponding secondary antibody (goat anti-rabbit IgG: 1 : 4000; BF03008, Biodragon-immunotech, Beijing, China; goat anti-mouse IgG: 1 : 4000, BF03001, Biodragon-immunotech, Beijing, China) for 2 h. Finally, the immunoblots were visualized with an ECL kit (CWBiotech, Beijing, China). The optical density of phosphorylated proteins (p-AKT, p-Rps6kb1, p-Eif4ebp1, and p-Yap) was measured by ImageJ software (National Institutes of Health, Bethesda, MD, USA) and normalized by the amount of the loading control on the same membrane.

The GMEC was plated and transfected for 48 h. Then, cells were lysed and total proteins were collected using RIPA reagent (R0010, Solarbio, Beijing, China) containing protease inhibitor cocktail. The concentration of proteins was examined with the BCA assay reagent. Detailed Western blot procedures were mentioned previously [38 (link)]. The primary antibodies included goat anti-PGC1α (ab106814, Abcam, Cambridge, UK; 1:500), rabbit anti-PPARγ (16643-1-AP, Proteintech Group, Wuhan, China; 1:2000), and mouse anti-β-Actin (CW0096, CW Biotech, Beijing, China; 1:2000). The secondary antibodies contained goat anti-mouse and anti-rabbit IgG (CW0102, 0103, CW Biotech, Beijing, China; 1:4000) and rabbit anti-goat-IgG (D110117, Sangon Biotech, Shanghai, China; 1:4000). After washing three times with TBST, the signals of protein were examined with a chemiluminescent (ECL) system. The content of protein was measured using Image J and normalized by comparison to β-Actin.