Western Blotting for Protein Expression of the Keap1/Nrf2/HO-1 Pathway and NF-κB Pathway

Total proteins were extracted using standard methods. The protein concentration was measured using a Bicinchoninic Acid Protein Assay Kit (batch number: 00171509; Kangwei Century Biotechnology, Beijing, China). Subsequently, 40 µg of protein was loaded onto 8 or 12% gels for sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Proteins were transferred onto polyvinylidene fluoride (PVDF) membranes. After transfer, membranes were blocked with 5% bovine serum albumin (Sigma–Aldrich) in 1× TBST (Tris-buffered saline and Tween-20) buffer at room temperature for 2 h. PVDF membranes were incubated with primer antibody at 4°C overnight, and then secondary antibody at room temperature for 2 h according to manufacturer instructions. Fluorescence signals were measured and analyzed using Odyssey Imager (LI-COR Biosciences, Lincoln, NB, USA). ImageJ 1.41 (NIH) was used for calculation of absorbance.

The main antibodies used in the experiments were supplied from Cell Signaling Technology: Keap1 (batch number: 8047S1901), Nrf2 (12721S1903), HO-1 (70081S1901), NF-κB p65 (8242S1902), phospho-NF-κB p65 (3033S1901), IκBα (4812S1902), phospho-IκBα (2859S1901), IKKα (2682S1902), phospho-IKKα (2697S1902), and β-actin (3700S1901).

To investigate the effect of SGF on activation of NF-κB p65, the proteins of nuclear fraction and cytoplasmic fraction were extracted with the NE-PER™ Kit (Thermo Scientific, Waltham, MA, USA). Western blotting was conducted as mentioned above, and PVDF membranes were incubated with primary antibodies. Histone H3 (batch number: 4499S2004; Cell Signaling Technology was the reference protein for the cell nucleus. The rinse and fluorescence signals analysis of membranes were performed as mentioned above.