Keap1-Nrf2-HO-1 Signaling Pathway Evaluation

The protocol was modified based on publications [25 (link),26 (link)]. Proteins of cells were lysed and collected with RIPA lysis solution. Protein concentration was determined by the BCA method. The protein samples of each group were loaded in equal quantities and transferred to the polyvinylidene fluoride membrane (BD Biosciences, USA) after electrophoresis. The membrane was blocked with QuickBlock™ blocking buffer (Beyotime) at room temperature and incubated overnight with a specific primary antibody for Keap1, Nrf2, HO-1, and β-actin (#ab227828, #ab137550, # ab13243, and #ab115777, Abcam, UK) at 4°C. TBST was used for a full washing, and the corresponding secondary antibody was added and incubated at room temperature for 1 h. After washing, ECL luminescent solution (Millipore, USA) was dropped to visualize protein bands. β-actin served as the internal reference.