Nuclear and cytoplasmic protein extraction kit

The Mouse Peritoneal Macrophage Isolation Kit was purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). An Enzyme-Linked Immunosorbent Assay (ELISA) Kit for evaluating PGE₂ was purchased from Abcam (Cambridge, Cambs, UK). The Mouse TNF-alpha ELISA Kit was purchased from eBioscience (San Diego, CA, USA). The Nuclear and Cytoplasmic Protein Extraction Kit was purchased from TransGen Biotech (Beijing, China).

Total protein was extracted with RIPA lysate, and cytoplasmic/nuclear proteins were extracted using the Nuclear and Cytoplasmic Protein Extraction Kit (TransGen Biotech). After determining the concentration by the BCA method, proteins were heated at 99°C for 10 min, subjected to SDS‐PAGE gel electrophoresis, and transferred to the PDVF membrane. Immunoblots were carried out with primary antibodies, including anti‐NFAT5 (Abcam, ab3446), anti‐AURKB (Abcam, ab2254), anti‐Phospho‐Ser/Thr (Abcam, ab17464), anti‐AQP4 (Abcam, ab2254), anti‐GAPDH (ZENBIO, 20030–67E4), anti‐Histone 3 (CST, 4499s).
Co‐IP assay was performed using a Direct Magnetic IP/CO‐IP Kit (Thermo Fisher Scientific, 88 824). Briefly, rat SDH tissue was lysed with IP lysis buffer supplied with protease and phosphatase inhibitors, and then incubated with the primary antibodies: anti‐NFAT5 (Novus Biologicals, NB1203446), or anti‐AURKB (Abcam, ab2254), or anti‐IgG (Proteintech, B900610). Magnetic beads (Thermo Fisher Scientific, 88 824) were used to pull down the corresponding protein complex. After eluted the proteins from the beads, and western blot was used to identify the specific protein.

After treated with 50 μg/mL of LMWF, DCs were collected at different time points (0, 30, 60, 120, and 240 min) to extract proteins using the Nuclear and Cytoplasmic Protein Extraction Kit (Beijing TransGen Biotech). Then, the protein concentrations were analyzed by the BCA Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Equal amounts of protein from cell extracts were loaded and electrophoresed on a gel (SDS-PAGE). After SDS-PAGE, proteins were transferred to PVDF membrane, and then blocked in 5% skimmed milk powder in a 37 °C incubator for an hour. After blocking, PBST (PBS-containing 0.05% Tween-20) was used to wash the membrane three times, 15 min for each wash. Then, the membrane was incubated with anti-β-actin (1:1000), JNK (1:1000), p-JNK (1:1000), p38 (1:1000), p-p38 (1:1000), NF–κB p65 (1:500), p-NF–κB p65 (1:500), iκi (1:500), p-iκ1 (1:500) ERK (1:1000), or p-ERK (1:1000) (Cell Signaling Technology, Danvers, MA, USA) at 4 °C overnight, washed three times, and incubated for an additional hour with corresponding peroxidase-conjugated HRP (1:1000) labeled secondary antibodies (Beyotime Biotech Co., Ltd. Shanghai, China) at 37 °C. After that, the membrane was washed three times. Finally, the membrane was viewed on Las 4000 (FVJIFILM corporation, Tokyo, Japan).