Anti p65

Cells from the various treatment groups were fixed with 4% paraformaldehyde at 4°C for 1 h and were permeabilized with 0.5% Triton X-100 for 10 min. After blocking with 1% bovine serum albumin (BSA; Beyotime Institute of Biotechnology) at 4°C for 30 min, the cells were incubated with anti-P65 (cat. no. 8242S; 1:100; Cell Signaling Technology, Inc.) at 4°C overnight. Subsequently, samples were incubated with DyLightFluor^® 488-conjugated donkey anti-rabbit IgG secondary antibodies (1:400; cat. no. BYE026; Shanghai Boyun Biotech Co., Ltd.) for 1 h at room temperature and were stained with DAPI for 7 min in the dark. The representative images were captured using an Olympus BX51 microscope (Olympus Corporation).

Immunofluorescence staining for p65 and LC3B-II was performed to precisely evaluate their expression in the cells. Following treatment, the cells were washed with ice-cold PBS and fixed in ice-cold acetone for 15 min. The cells were then blocked in 10% normal goat serum (cat. no. C0265; Beyotime Institute of Biotechnology, Co., Ltd.) at 37°C for 1 h, following which they were washed by PBS and incubated at 37°C with anti-p65 (1:200; cat. no. 710048) or anti-LC3B-II antibody (1:200; cat. no. 700712) (both Thermo Fisher Scientific, Inc.) for 1 h. Following three washes, the cells were incubated with FITC-conjugated goat anti-rabbit secondary antibodies (1:200; cat. no. A0562; Beyotime Institute of Biotechnology, Co., Ltd.) for 1 h at 37°C. The slides were mounted with 4′,6-diamidino-2-phenylindole (DAPI). Confocal images were captured (26 (link)) using a confocal microscope (Olympus Corp., Tokyo, Japan) at the excitation and emission wavelengths of 495 and 517 nm for FITC, 649 and 680 nm for Cy5, and 358 and 463 nm for DAPI nuclear staining, respectively.