Hrp goat anti mouse igg h l

The cells were collected and RIPA lysis buffer (Meilun Biotechnology Co., Ltd., Suzhou, China) containing PMSF (1 mM) (Meilun Biotechnology Co., Ltd., Suzhou, China) was added for protein extraction. Total protein concentration of samples was detected by BCA protein assay. After diluting the protein sample to the same concentration with RIPA lysis solution, added 5 × loading buffer, boiled in 100 °C water bath for 5 min, and store at -80 °C after cooling on ice. 30 μg total protein samples were taken from each group, separated by SDS-PAGE gels electrophoresis and transferred to PVDF membranes. After sealing with 5% bovine serum albumin (Albumin from bovine serum, BSA), the membranes were incubated with primary antibodies anti-VEGF (1:2000, 66,828–1-IG; proteintech), anti-HIF-1α (1:2000, 66,730–1-IG; proteintech), or anti-GAPDH (1:2000, 60,004–1-IG; proteintech). Following incubation with the secondary antibody solution of HRP Goat Anti Mouse IgG (H + L) (1:5000, proteintech). The PVDF membranes were color-coded by chemiluminescence method and luminescence detection by gel imaging system Versa DocTM Imaging System to collect strip images. GAPHD was used as internal reference to calculate the relative expression levels of each target protein by Image Lab analysis software (National Institutes of Health).

Penfluridol (purity ≥ 98.92%) was obtained from MedChemExpress (HY-B1077, Shanghai, China). Lipopolysaccharides (LPS from Escherichia coli 055: B5) was acquired from Sigma Chemical Co. (St. Louis, MO, USA). High-glucose DMEM and FBS were obtained from Solarbio (Beijing, China). Enhanced Cell counting Kit-8 (CCK-8) was purchased from Bioss (Beijing, China). Mouse and human ELISA kits for TNF-α, IL-6, IL-18, and IL-1β were acquired from BOSTER Biological Technology Co. Ltd., (Wuhan, China). The nitric oxide (NO), malondialdehyde (MDA), and superoxide dismutase (SOD) kits were obtained from Beyotime Institute of Biotechnology (Beijing, China). Primary antibodies against NLRP3, cleaved Caspase-1 and Nrf2 were purchased from Cell signaling. Antibodies against HO-1, lamin B, GAPDH, and β-tublin were purchased from proteintech Group, Inc (Wuhan, China). Primary antibody against IL-1β was purchased from Abcam (ab234437; Abcam, Cambridge, UK). HRP-Goat Anti-Mouse IgG (H + L) and HRP-Goat Anti-Rabbit IgG (H + L) were purchased from proteintech group, Inc (Wuhan, China).

Proteins were separated by SDS-PAGE. After electrophoresis, gels were cut and transferred to PVDF membranes. Membranes were blocked with 5% w/V evaporated milk in PBST. Diluted primary antibodies were added and incubated overnight at 4 °C. Primary antibodies were against TRIM36 (1:1000, Sigma), BAX (1:10,000, Proteintech), and BCL2 (1:2000, Proteintech), caspase-3 (1:1000, Abclone), cleaved caspase-3 (1:200, Sigma), caspase-7 (1:5000, Proteintech), PARP1 (1:1000, Abclone), MMP-9 (1:500, Proteintech), cyclin D1 (1:1500, Proteintech), β-catenin (1:20,000, Proteintech), active β-catenin (1:1000, Cell signaling technology), c-JUN (1:2000, proteintech), Histone H3 (1:10,000, Proteintech), Actin (1:10,000, Proteintech). Thereafter, membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (H + L) or HRP-goat anti-mouse IgG (H + L) secondary antibodies (Proteintech) for 50 min at room temperature. Chemiluminescence was measured using Super Signal West Atto (Thermo Fisher Scientific). Blots were imaged with Amersham Imager 600 software and analyzed using ImageJ software.