Rabbit anti nrf2 monoclonal antibody

Immunoprecipitation was performed per the standard protocol as previously reported (Yang et al., 2018 (link)). Astrocytes were treated according to experiment designs, which were lysed with immunoprecipitation buffer and centrifuged at 12000 g for 10 min at 4°C. The supernatant was gathered for co-immunoprecipitation (Co-IP) and estimated for protein concentration. Then the lysates were pre-clearing with 20 μL washed protein A/G agarose (Santa Cruz Biotechnology) and centrifuged briefly to collect the supernatant. Lysates (1 mg protein) was incubated with 1 μg rabbit anti-Nrf2 monoclonal antibody (Santa Cruz Biotechnology), or 1 μg normal rabit IgG (Santa Cruz Biotechnology) with shaking for 12 h at 4°C. 20 μL protein A/G agarose was added to the complex and shaken for 4 h at 4°C. The agarose was then collected via centrifugation. Twenty μL 2^∗loading buffer was added to the agarose bounding to the protein, boiled together at 99°C for 5 min, followed by WB analyzing with mouse anti-Cx43 monoclonal antibody (Invitrogen) to assess the connection between Cx43 and Nrf2 protein.

Lung tissues were snap frozen in liquid nitrogen, pulverized and resuspended in ice-cold lysis buffer (Solarbio, Beijing, China). Protein concentrations were determined with the Bradford method. Lysates were allowed to solubilize on ice for 30 min, and particulate mass was removed by centrifugation at 15,000 × g for 15 min at 4°C. Supernatants were analyzed by SDS-PAGE. Primary antibodies used included rabbit anti-Tollip monoclonal antibody (1:400), rabbit anti-NF-κB monoclonal antibody (1:400), rabbit anti-IRAK1 monoclonal antibody (1:400), rabbit anti-TLR4 monoclonal antibody (1:400), rabbit anti-TRAF6 monoclonal antibody (1:400), rabbit anti-VEGF-α monoclonal antibody (1:400), rabbit anti-Nrf2 monoclonal antibody (1:400), mouse anti-GAPDH monoclonal antibody (1:400) were purchased from Santa Cruz Biotechnology, Inc.. Secondary antibodies were horseradish peroxidase-labeled antibodies (Thermo Scientific Pierce, Rockford, IL, USA). Blots were processed for enhanced chemifluorescence using a Pierce ECL Western blotting substrate (Thermo Scientific Pierce).