Ab104135

To visualize the nuclear translocation of NF-κB in ALL-SIL cells, isogenic control and venetoclax-resistant cells were plated on poly-L-lysine coated coverslips placed in a 6-well plate. After 12 to 16 hours, the cells were fixed for 15 minutes with 4% paraformaldehyde at room temperature and washed three times with PBS. Then they were blocked in 5% goat serum with 1 mg/mL BSA and 0.3% TritonX-100 for 2 hours. Rabbit anti-human NF-κB p65 antibody (D14E12, Cell Signaling Technology) was used to stain cells overnight at 4°C. The following day, cells were washed three times with PBS for 5 minutes, and A488-conjugated goat anti–rabbit secondary antibody (Invitrogen) was applied for 1 hour at room temperature. After secondary antibody incubation, cells were washed three times with PBS for 5 minutes each and counterstained with DAPI for 5 minutes. Afterward, cells were washed three times with PBS for 5 minutes each. The coverslips were mounted using anti-fade fluorescence mounting medium (Abcam, ab104135), and cells were imaged using a Leica TCS SP8 Confocal/Multi-Photon high-speed upright microscope. Nuclear to cytoplasmic NF-κB p65 ratio was calculated using ImageJ.