Nonidet p 40 np 40 lysis buffer

An IP assay was used to explore the acetylation level of FoxO3a. Fresh mouse heart tissue or H9c2 cells were lysed with non‐denaturing Nonidet P‐40 (NP‐40) lysis buffer (Beyotime) supplemented with protease and phosphatase inhibitor cocktail (Abcam). An amount of 500 μg protein was immunoprecipitated with anti‐FoxO3a antibody and PureProteome protein A/G mix magnetic beads (Merck Millipore) overnight at 4°C. Beads coated with the immune‐precipitated protein complex were washed and denatured at 95°C for 10 minutes. The immunoprecipitated proteins were then subjected to SDS‐PAGE. The antibodies used were as follows: FoxO3a (1:50; Cell Signaling Technology, #12829) and acetylated lysine (1:1000; Cell Signaling Technology, #9441).

Cells were lysed in nonidet-P40 (NP40) lysis buffer (Beyotime) supplemented with protease and phosphatase inhibitors. The protein concentrations were determined using the BCA protein assay kit (Thermo Fisher). The cell lysates were boiled in SDS loading buffer. The samples were subjected to SDS-PAGE and transferred to a nitrocellulose membrane. The membranes were incubated with primary antibodies overnight at 4 °C. After being washed in TBST and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies, the membranes were visualized using enhanced chemiluminescent (ECL) substrates.