Ab153914

Samples were lysed using radioimmunoprecipitation assay buffer (RIPA) lysis buffer (150 mM NaCl, 5 mM EDTA, 50 mM Tris pH 8.0, 0.5% sodium deoxycholate, 1% NP-40, 0.1% SDS) supplemented with 1 mM dithiothreitol and protease inhibitors. The lysates were separated by SDS-PAGE and analyzed using standard western blotting procedures (Khan et al., 2019 (link)). Antibodies: human NR4A1 (ab153914, 1:1,000; Abcam), mouse NR4A1 (14-5965-82, 1:1,000; Invitrogen), NR4A2 (AV38753, 1:1,000; Sigma-Aldrich), NR4A3 (TA804893, 1: 1,000; Thermo Fisher Scientific), VHL (68547, 1:1,000; Cell Signaling), β-actin (13E5, 1:5,000; Cell Signaling), β-tubulin (9F3, 1: 5,000; Cell Signaling), and GAPDH (D16H11, 1: 5,000; Cell Signaling) were employed for protein detection.

Livers were fixed with 4% formalin for 24 h and subsequently embedded in paraffin and cut into 5-μm-thick sections. For liver histopathology, samples were stained with hematoxylin and eosin (H&E). For immunohistochemical (IHC) staining, samples were dehydrated, exposed to antigen, and then incubated with IL-1β (ab283818, Abcam, 1:100 dilution), Caspase 6 (ab185645, Abcam, 1:100 dilution), and Ly-6G (ab261916, Abcam, 1:100 dilution) antibodies respectively at 4 °C overnight. For immune-fluorescence (IF) staining, tissue sections or cultured cells were fixed with 4% formalin for 30 min and then incubated at 4 °C overnight with antibodies against CD11b (ab184308, Abcam, 1:100 dilution), CD68 (#26042, Cell signaling Technology, 1:100 dilution), Caspase 6 (ab185645, Abcam, 1:100 dilution), NR4A1 (ab153914, Abcam, 1:100 dilution); SOX9 (ab185966, Abcam, 1:100 dilution), NEK7 (sc-393539, Santa Cruz Biotechnology, 1:100 dilution), and NLRP3 (ab270449, Abcam, 1:100 dilution). Then samples were incubated with the secondary antibody conjugated to Alexa Fluor 488 (Jackson Immunoresearch) or Alexa Fluor Cy5 (Jackson Immunoresearch) for 2 h at room temperature in the dark. Immunofluorescence images were captured using a fluorescence microscope (Keyence BZ-X810, Osaka, Japan).