Protein Extraction and Immunoblotting Protocol

The total protein was extracted using RIPA lysate (#R0278, Sigma) according to the manufacturer's instructions. The BCA protein assay kit (Beyotime, Shanghai, China) was used to measure the protein concentration according to the manufacturer's instructions. An equal amount of protein was loaded onto sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and immunoblotting was performed as described previously [24 (link)]. Primary antibodies against HO-1 (1 : 1000), Nrf2 (1 : 1000), Bax (1 : 1000), Bcl-2 (1 : 1000), NF-κB p65 (1 : 1000), c-PARP (1 : 1000), GAPDH (1 : 2000), phosphor-NF-κB p65 (1 : 1000), p-IκB-α (1 : 1000), and IκB-α (1 : 1000) were used. HRP-conjugated secondary antibodies were used to detect fluorescence using BeyoECL Plus as described previously [24 (link)].