Ab72520

When the A549/DDP cells were grown until the cell confluence reached approximately 70%, the fixation and staining treatment are started. After that, added primary antibody overnight. The first antibody was anti-CHAF1B antibody (1:200, ab72520 Abcam) and NCOR2 (1:100, ab24551abcam). The cells were then incubated with Alexa Fluor^® 488 Goat Anti-Mouse antibody labeled diluted 1:100 in blocking solution. Nuclei were stained with DAPI for 5 min. Cells were examined with LeicaTCS-SP5 laser confocal scanning microscope.

Tissue derived from animal experiments and clinical specimens. Firstly, sections were prepared, dewaxed, hydrated. Then, paraffin sections were repaired with tissue antigen. The sections were treated with 50 μl peroxidase to block the endogenous peroxidase activity for 10 min. Subsequently, the sections were probed with polyclonal anti‐Ki-67 (1:100; GTX16667, genetex), anti‐Active-Caspase 3 (1:200; ab2302, abcom) and CHAF1B (1:500; ab72520, Abcam) at 4 °C overnight. The bound antibodies were detected with a biotin-labeled second antibody, diaminobenzidine was added for visualizing, and hematoxylin was used as a counterstain. Finally, the sections were dehydrated and cleared and sealed with neutral gum. Two pathologists who were blinded without knowledge of the clinical or histopathological data scored the samples. The score was determined by the proportion of positive tumor cells and the degree staining intensity [20 (link)].

The protein in A549 and A549/DDP and tissues was extracted by Radioimmunoprecipitation assay buffer (RIPA) (Auragene, Changsha, China). Proteins were quantified by bicinchoninic acid (BCA) protein assay kit (Thermo Scientific). Proteins were separated on a 10% SDS-PAGE gel and transferred onto nitrocellulose membranes, and then incubated with 5% BSA for 2 h at 25 °C. The membranes were incubated with Anti-CHAF1B antibodies (1:500; ab72520, Abcam); Anti-NCOR2 antibodies (1:500; ab24551, Abcam); Anti-PPP5C antibodies (1:500; sc271612, Santa Cruz) was incubated overnight at 4 °C. Then it was incubated with a secondary HRP-labelled goat anti-rabbit IgG antibody (1:4000; Abcam) for 1 h at room temperature. Immunoassay were performed by ECL combined with Western Blot system (Auragene, Changsha, China). GAPDH (1:4000; ab125247, Abcam) was used as the internal control of Western blotting.