Anti nrf2

Protein extraction was performed by using Cell Lytic buffer (Sigma-Aldrich) supplemented with a protease inhibitors cocktail (Sigma-Aldrich) plus phosphatases inhibitors (Na3VO4 1 mM; NaF 10 mM) and resolved by electrophoresis through NuPAGE Bis-Tris gel (Invitrogen) and electroblotted onto nitrocellulose (Protran, Sigma-Aldrich) membrane. Blots were incubated with indicated primary antibodies in 5% non-fat dry milk in PBS plus 0.1% Tween20 overnight at 4 °C. Primary antibodies were: anti-PERK (1:500; Cell Signaling; Danvers, MA, US), anti-ERK1/2 (1:500; Cell Signaling), anti-ERK1/2 (1:500; Cell Signaling), anti-Nrf2 (1:500; Genetex; Alton Pkwy Irvine, CA, US), anti-Herp (1:500; Sigma-Aldrich), anti-AKR1C1 (1:500; NOVUS), anti-AKR1C2 (1:500, Merck), anti-AKR1C3 (1:500; Cell Signaling), anti-Tubulin-α (1:5000; Santa Cruz Biotechnology; Santa Cruz, CA, US). Detection was achieved using horseradish peroxidase-conjugate secondary antibody (1:5000; Jackson ImmunoResearch; Cambridge, UK) and visualized with ECL plus (Amersham Biosciences; Amersham, UK). Images were acquired by using a ChemiDoc™ Touch Imaging System (Bio-Rad; Berkeley, CA, US) and analyzed by Image Lab software (Bio-Rad).

Maresin‐1 and Maresin‐1 ELISA kit were purchased from Cayman Chemical. AngII was purchased from Enzo Life Sciences. Mouse IL‐1β ELISA kit was purchased from Neobioscience. EdU imaging kits was purchased from APExBIO. ML385 and KN‐93 were purchase from Topscience. Fluo‐4 AM was purchased from Invitrogen. Fetal bovine serum was purchased from Inner Mongolia Opcel Biotechnology Co., Ltd. Primary antibodies included: anti‐GAPDH (Abcolonal), anti‐SM22α (Servicebio), anti‐ASC (Santa Cruz Biotechnology), anti‐caspase‐1 p20 (Santa Cruz Biotechnology), anti‐IL‐1β (Santa Cruz Biotechnology), anti‐OPN (Immunoway), anti‐NLRP3 (Immunoway), anti‐IL‐18 (ImmunoWay), anti‐GSDMD‐N (Immunoway), anti‐Nrf2 (GeneTex), anti‐α‐SMA(Abcam), anti‐HO‐1(Abcam), anti‐CaMKII (Abcam), anti‐p‐CaMKII (Zenbio), anti‐PCNA (Cell Signaling Technology), anti‐rabbit IgG (H+L) (Cell Signaling Technology), anti‐mouse IgG (H+L) (Cell Signaling Technology), Cy3‐conjugated goat anti‐rabbit IgG (H+L) (Servicebio), and Alexa Fluor® 488‐conjugated goat anti‐rabbit IgG (H+L) (Servicebio).

Cell lysates were prepared using M-PER (Thermo Fisher Scientific, Waltham, MA, USA) buffer with protease inhibitor cocktail set III (Merck, Darmstadt, Germany). The contents of proteins in the lysates were determined using Bradford reaction. Samples (40 μg) were solubilized in Laemmli buffer added with 2% mercaptoethanol (Bio-Rad, Hercules, CA, USA) and were then subjected to 10% SDS (sodium dodecyl sulfate)-polyacrylamide gel electrophoresis as described earlier [21 (link)]. Following transfer, membranes were blocked for 1 h at room temperature in the presence of casein in TBS (tris-buffered saline)–1% Tween buffer (Bio-Rad) and subsequently incubated overnight at 4 °C with the following primary antibodies: anti-COX-2, anti-Nrf2, anti-FABP₄, anti-β-actin (GeneTex Inc., Irvine, CA, USA), and anti-PPARγ (Cayman Chemical, Ann Arbor, MI, USA), all of which were diluted to the ratio of 1:1000. After incubation, the membranes were washed and incubated with secondary antibodies (anti-rabbit IgG (HRP); Thermo Fisher Scientific, Waltham, MA, USA) for 1 h at room temperature. Then, the membranes were washed again, and proteins were detected using a Clarity Western ECL Luminol Substrate detection kit (Bio-Rad). The integrated optical density of the protein bands was quantified using a Chemi Doc Camera with Image Lab software (Bio-Rad).