Af5393

Differentially expressed proteins such as HMOX1, MP2K5, XRCC1 and A1AG were measured by Western blot. Human lung AEC-II cells (Lanmeng Biotechnology, Hebei, China) were harvested after the treatments. After lysis for 30 min on ice with Cell Protein Extraction Reagent, the total protein concentration was determined by BCA assay according to the manufacturer's instructions. Each sample containing approximately 50 μg of proteins was electrophoresed on 12% SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked with 5% non-fat milk for 2 h at room temperature. Primary antibodies (DF6623, AF5393, AF0296, DF7667, Affinity Biosciences, OH, USA) were incubated at 4 °C overnight. After washing, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (ZB-2306, ZB-2305, ZSGB-BIO, Beijing, China). The immunoreactive bands were detected with an enhanced chemiluminescent (ECL) reagent kit according to the manufacturer's protocol and a chemiluminescence detection system. The obtained images were analyzed by Image J software to analyze the gray value of the target bands for relative quantitative information analysis of the proteins.

Brain samples were collected on day 7 after ICH. Brain tissues or cell samples were washed with PBS and permeabilized with 0.1% Triton X-100. Samples were incubated with rabbit anti-HO-1 (1:200, AF5393, Affinity Biosciences) and mouse anti-Nrf2 (1:200; AF0639, Affinity Biosciences) at 4°C overnight. Following PBS washes, the samples were incubated with Alexa Fluor-conjugated secondary antibodies (anti-rabbit 488 or 594, 1:200, A32766, A48284, ThermoFisher Scientific, MA, USA) for 1 hour at room temperature, followed by incubation with 4′,6-diamidino-2-phenylindole (DAPI) for 2 minutes. Images were observed with a fluorescence microscope (Leica DM2500, Leica Co.) and analyzed using ImageJ software (NIH, Bethesda, MD, USA). The allocation signals in the dual-channel overlay images were analyzed with the ImageJ “Co-localization Finder” plugin.

After treatment, the cells were harvested and lysed using a cell buffer (WB038, GEFAN, Nanjing, China), and their concentrations were estimated by a BCA kit (GM03, GEFAN). After denaturation, cells were subjected to protein electrophoresis. After transferring to a PVDF membrane (WB041, GEFAN), the membrane was sealed with 5% skim milk. After 2 h, cells were reacted with primary antibodies overnight at 4°C followed by incubation with a goat anti-rabbit secondary antibody IgG H&L (1:3000, S0001, Affinity Biosciences, Cincinnati, OH, USA) for 1 h at 37°C. Finally, a color reagent (E266188, Aladdin, Shanghai, China) was used to visualize the bound antibodies, which were then placed in a chemiluminescent instrument (610020-9Q, Clinx, Shanghai, China). The primary antibodies of the rabbit anti-human Nrf2 (1:2000, AF0639), SOD-1 (1:1500, AF5198), CAT (1:2000, AF7746), HO-1 (1:1500, AF5393), nuclear factor kappa B (NF- κB, 1:1500, AF5006), phospho-IkappaB-alpha (p-IKBα, 1:2000, AF2002), and GAPDH (1:20000, AF7021) were obtained from Affinity Biosciences .

The protein sample was collected from liver tissues using a protein extraction kit (BC3710, Solarbio, China) or a Nuclear and Cytoplasmic Extraction Reagent (78833, Thermo Fisher, USA). The protein was quantified using a BCA kit (PC0020, Solarbio, China). After separating the protein by SDS-PAGE, the protein was transferred into a polyvinylidene fluoride membrane (10600023, GE Healthcare Life, USA). The membrane was blocked by 5% BSA for 1 hour, then the membrane was reacted with primary antibodies and secondary antibodies. The relative intensity of the protein bands was quantified using ana ECL kit (36208ES60, YEASEN, China) with the iBright FL1500 Imaging System (A44115, Invitrogen, USA). The results were counted by ImageJ2x (Rawak Software, Germany). The primary antibodies and secondary antibodies were as follows: anti-p53 (AF0879, Affinity, USA), anti-β-actin (AF7018, Affinity, USA), Nrf2 (AF0639, Affinity, USA), Lamin B (DF6687, Affinity, USA), Heme oxygenase-1 (HO-1, AF5393, Affinity, USA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, AF7021, Affinity, USA), and goat anti-rabbit (S0001, Affinity, USA).

Cells were harvested and lysed in ice-cold RIPA (Radio-Immunoprecipitation Assay) buffer mixed with cocktail and PMSF for 30 min. The BCA protein assay kit (BB-3401, Shanghai Beibo Biotechnology, Shanghai, China) was used to measure the protein concentration. Then, equal amounts of protein were resolved by 7.5% to 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene fluoride (PVDF) membranes.
After blocked with 5% skim milk for 2 h at room temperature, the membranes were incubated with appropriate primary antibodies against p-NF-κB p65 (CST, #3033S, 1:1000), NF-κB p65 (CST, #8242S, 1:1000), p-IKB-α (GeneTex, #32224, 1:1000), IKB-α (GeneTex, #110521, 1:1000), NLRP3 (Abcam, #ab263889, 1:1000), ASC (Affinity, #DF6304, 1:500), IL-1β (Affinity, #AF5103, 1:1000), caspase-1 (Affinity, #AF5418, 1:1000), Nrf2 (Affinity, #AF0639, 1:1000), HO-1 (Affinity, #AF5393, 1:1000) and β-actin (Affinity, #AF7018, 1:1000) overnight at 4°C. Subsequently, membranes were incubated with secondary HRP-conjugated goat anti-rabbit IgG (Immunoglobulin G, Affinity, #072102, 1:5000) for 1 h. Protein bands were visualized using the enhanced chemiluminescence (ECL) (Tanon 4200SF, China) detection system. The intensity of the bands was assessed using the ImageJ software (National Institutes of Health, Bethesda, MA, USA) tool.