Goat anti rabbit

The homogenate of liver tissue or cells was prepared in lysis buffer (20 mM Tris–Cl pH7.5, 140 mM NaCl, 1 mM CaCl₂ and MgCl₂, 10 mM NaF, 1% NP-40, 10% glycerol, 2 mM Na-Vanadate, and 1 mM PMSF) supplemented with complete Protease Inhibitor Cocktail (cOmplete™, Sigma-Aldrich, Dallas, TX) for 30 min, centrifuged at 12,000 rpm at 4°C for 15 min. The protein samples were resolved by SDS-polyacrylamide gel electrophoresis and electrophoretically transferred to PVDF membranes. The membranes were blocked at room temperature for 2 h in 5% defatted milk dissolved in TBST (10 mM Tris–HCl, pH 7.4, and 150 mM NaCl), incubated at 4°C for overnight with the following antibodies: PPARγ (#AF6284, Affinity, 1:1000), FGF9 (#A6374, ABclonal, 1:500 and #ab206408, Abcam, 1:500), ChREBP (#A7630, ABclonal, 1:1000), Fasn (#DF6106, Affinity, 1:1000), or β-Actin (#AF7018, 1:10000), washed in TBST (0.1%Tween 20) for 15 min (repeated three times) and incubated with a goat anti-mouse (# S0002, Affinity) or goat anti-rabbit (# S0001, Affinity) IgG (H + L) HRP secondary antibody (1:5000 dilution in TBS) for 1 h at RT. Immunoreactivity was visualized and quantified by infrared scanning using the Odyssey system (LI-COR Biosciences).

The Mori Fructus fruits (Morus nigra Linn.) samples were sourced from Guangxi Province (China), 30% hydrogen peroxide was bought from Chengdu Keen (Chengdu, China), ROS, SOD, Gpx, BCA protein quantification and plasmatic nucleus isolation protein extraction kits are all from Beyotime Institute of Biotechnology (Shanghai, China). Cell Counting Kit-8(CCK-8) bought from Everbright, MEM medium, phosphate buffer and NQO1 antibodies were obtained from Boster (California, America). Lamin B and Nrf2 antibodies were from Proteintech (Wuhan, China), HO-1, Gapdh, AKT, p-AKT p-PI3K and PI3K antibodies were all obtained from Affinity(America), goat anti-mouse from Beyotime and goat anti-rabbit from Affinity.

The levels of USP13 and NLRP3 in brain tissues and SV‐HUC‐1 cells were assessed by immunofluorescence. Brain tissues were removed, fixed in 4% paraformaldehyde solution for 24 hours at 4°C. After embedding in paraffin, the tissues were cut into 4‐µm‐thick continuous coronal brain slices. Brain tissues and cells were blocked with 5% BSA blocking solution for 60 minutes at room temperature, followed by washing with PBS. Samples were then incubated with USP13 antibody or NLRP3 antibody at 4°C overnight. Sections were washed 3 times (5 minutes each time) with ice‐cold PBS and further incubated with goat anti‐rabbit IgG‐AF488 (Affinity, USA) secondary antibody.
Cells were fixed with 4% paraformaldehyde for 20 minutes and permeabilized with 0.5% Triton X‐100 for 20 minutes. Cells were blocked for 60 minutes at room temperature using 5% BSA blocking solution and then washed with PBS. Thereafter, cells were incubated with 1:200 dilution of primary antibodies (rabbit anti‐NLRP3 antibody, Affinity, USA) for overnight at 4°C. Following overnight incubation, cells were washed three times with PBS and incubated for 2 hours in dark with second antibody (goat anti‐rabbit, Affinity).
Later, the nuclei of cells were stained with 4′,6‐diamidino‐2‐phenylindole (DAPI, C0060, Solarbio); representative fluorescence images were obtained using fluorescence microscope (BX63, Olympus).