Rabbit anti ho 1 antibody

Total protein was extracted from brain tissues with a SDS lysis buffer (Beyotime, Shanghai, China), supplemented with 1% phenylmethylsulfonyl fluoride (Beyotime, Shanghai, China). Equal amount of proteins was analyzed by 10% SDS–PAGE and transferred to PVDF membranes. After being blocked in 5% non-fat milk at room temperature for 1 h, the membranes were incubated with primary antibodies at 4°C overnight, including rabbit anti-COX-2 antibody (Cell Signaling, United States), rabbit anti-TNF-α antibody (Millipore, United States), rabbit anti-IL-1β antibody (Abcam, United States), rabbit anti-p-NF-κB (Cell Signaling, United States), rabbit anti-TRAF6 antibody (Proteintech group, United States), mouse anti-IRAK1 antibody and rabbit anti-Nrf2 antibody (Santa Cruz Biotechnology, United States), rabbit anti-HO-1 antibody (Abcam, United States), and rabbit anti-β-actin antibody (Cell Signaling, United States). The appropriate peroxidase-conjugated antibodies, anti-mouse, or anti-rabbit were incubated with the membranes at room temperature for 2 h. Signals were detected using ECL-Plus (Merck Millipore, Darmstadt, Germany) and quantified using a Bio-Rad 2000 gel imaging system with QUANTITY ONE software (Bio-Rad Laboratories, Hercules, CA, United States).

Total protein was extracted from mouse lungs and cells by RIPA. Protein concentration was detected by the bicinchoninic acid assay (BCA, Thermo Fisher Scientific). Equal amounts of protein (30 µg) were loaded on SDS-PAGE and transferred to a polyvinylidene fluoride membrane (Zhou et al. 2016 (link)). After blocking with skim milk (5%) in Tris-buffered saline (TBST) for 1 h at room temperature, the membrane was probed with the primary antibodies at 4 °C overnight: rabbit anti-NLRP3 antibody (1:2000; CST, Danvers, MA), rabbit anti-Nrf2 antibody (1:1000, CST), rabbit anti-HO-1 antibody (1:1000, ABCAm), goat anti‐IL‐1β antibody (1:2000; R&D, Minneapolis, MN), rabbit anti‐caspase‐1 antibody (1:1000; ABCAm, Eugene, OR), rabbit anti‐ASC antibody (1:1000, CST), rabbit anti-Fn14 antibody (1:1000, ABCAm), rabbit anti-β-actin antibody (1:7500, Signal way Antibody, College Park, MD, USA), and rabbit anti‐α‐tubulin antibody (1:7500, Servicebio, China). After incubation with peroxidase‐conjugated secondary antibodies (1:7500, Signal way Antibody) at room temperature for 1 h, the signal was developed using an ECL chemiluminescence kit (Millipore, USA). The band intensities were quantitated using Image Lab software and normalized to internal reference values accordingly.