4.4. Western Blotting

After 8 h of IL-25 treatment or pretreatment with the NAC, rotenone (Sigma-Aldrich, #R8875), antimycin A, AMPK, or Mdivi-1, THP-1 cells and THP-1 derived macrophages were lysed with equal volumes of ice-cold lysis buffer. Equal amounts of cell lysates were separated using SDS-PAGE and transferred to PVDF membranes. After blocking in PBS containing 5% milk and 0.1% Tween 20, the membrane was incubated with anti-phospho (p)-5′-AMP-activated protein kinase alpha (AMPKα, #2535), anti-AMPKα (#5832), anti-PINK1 (#6946), anti-GAPDH antibodies (#2118S) (all from purchased Cell Signaling Technology, Danvers, MA), anti-p-Parkin (Abcam, Cambridge, MA, #ab73015), anti-LC3 (GeneTex, CA, USA, #GTX127375), or anti-Parkin (Abcam, #ab77924) to further investigate the influence of IL-25 on mitophagy. Immunoreactive bands were visualized using a horseradish peroxidase-conjugated secondary antibody and an enhanced chemiluminescence (ECL) system (Merck Millipore, Darmstadt, Germany, #WBKLS0500). The assay was performed by using the protocols recommended by the manufacturer. After chemiluminescent detection of phosphoproteins, we used Gentle Review™ Stripping Buffer (VWR LIFE SCIENCE, Pennsylvania, USA, #N552) to strip the PVDF membrane. We added 10 mL stripping buffer to incubate the membrane with gentle sharking for 30 min at room temperature.