Mouse anti cx43 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.

Brain cryosections and astrocytes coverslips were performed as previously described (Song et al., 2019 (link)), and immunostained with following primary antibodies: rabbit anti-Nrf2 polyclonal antibody (1:200, Santa Cruz Biotechnology, United States), rabbit anti-HO-1 polyclonal antibody (1:300, Abcam, United Kingdom), mouse anti-Cx43 monoclonal antibody (1:200, Invitrogen, United States), rabbit anti-GFAP polyclonal antibody (1:1000, Servicebio, China), mouse anti-VIM monoclonal antibody (1:500, Servicebio). The secondary antibodies used (1:500, Beyotime): Alexa Fluor 488 goat anti-rabbit IgG, Alexa Fluor 488 goat anti-mouse IgG, Alexa Fluor 555 donkey anti-mouse IgG, Alexa Fluor 555 donkey anti-rabbit IgG, Alexa Fluor 647 goat anti-mouse IgG, Nuclei were stained with DAPI (1:5000, Beyotime). The fluorescence images were observed and analyzed by a confocal laser-scanning microscope (Leica).