2.4.5. Western blotting

The protein was extracted by Invent Biotechnologies, SM-005, and dissolved in denatured protein lysate (Invent Biotechnologies, WA-009). After high-temperature denaturation, the protein concentration was determined by BCA (Beyotime, P0012). An equal amount of protein from each experimental group was separated on 4–20% SurePAGE (Genscript, M00656) and transferred onto a 0.45-μm PVDF membrane (Millipore, IPVH00010). Then, 5% non-fat dry milk was sealed and then treated with the first antibody overnight at 4 °C. After cleaning the unbound first antibody, the membrane was treated with a second antibody (Epizyme, China) at room temperature for 1 h. Image Lab (Bio-Rad, California, USA) was used for imaging and quantitative analysis. The Stripping Buffer (APPLIYENP, 1650) was used to wash the antibody and re-incubate with the first antibody overnight, followed by imaging and analyses.