Ac nf κb

To assess the protein levels of Bcl-2, cleaved-caspase-3, Ac-FoxO1, Ac-NF-κB, and Ac-p53, 50 μg of total protein were subjected to SDS-PAGE and transferred onto PVDF membranes. Membranes were blocked with 5% non-fat milk at room temperature for 3 h, incubated with primary antibodies specific to Bcl-2 (1:1000, Abcam, Cambridge, UK), cleaved-caspase-3 (1:1000, Abcam), Ac-FoxO1 (1:1000, Abcam), Ac-NF-κB (1:1000, Cell Signaling Technology, Beverly, MA), Ac-p53 (1:1000, Cell Signaling Technology), BAX (1:1000, Cell Signaling Technology), SIRT1 (1:1000, Cell Signaling Technology), or β-actin (1:1000, Abcam) at 4 °C overnight. Then, membranes were incubated with HRP-conjugated secondary antibodies diluted at 1:3000 (Boster, Wuhan, China) at 37 °C for 1 h. Protein bands on the membrane were visualized with ECL Kit (Millipore, USA) using FluorChem FC system (Alpha Innotech). Results were presented as densitometric ratio between the protein of interest and the loading control (β-actin).

Overnight incubation of cell lysates proceeds with NF-κB or STAT3 antibodies (1:200) at 4 °C. Later, washed protein G beads were added and the mixture was incubated at 4 °C for 1 h. Subsequently, the lysis buffer was used to wash the immunoprecipitates for five times, and then the proteins were eluted with the aid of sodium dodecyl sulfate (SDS) sample buffer. Finally, the samples were loaded on SDS polyacrylamide gel electrophoresis (SDS-PAGE) gels and analyzed by western blotting using ac-NF-ΚB (1:1000, Cell signaling technology, 3045S), ac-STAT3 (1:1000, Cell signaling technology, 2523S) antibodies.