Sc 7392

HT-29 cells were plated on confocal dishes (100350, SPL). After treatment with the indicated chemicals, the cells were fixed with 4% paraformaldehyde for 10 min at room temperature. Cells were incubated with immunofluorescence blocking buffer (PBS with 3% BSA, 1% saponin, and 1% Triton X-100) containing anti-MYC (1:100; 5605, Cell Signaling) or anti-RIPK3 antibodies (1:50; 13526. Cell Signaling) overnight at 4 °C. Samples were washed with PBS three times and then incubated with Alexa Fluor 488 anti-rabbit (1:200; A-11008, Thermo Fisher Scientific) supplemented in immunofluorescence blocking buffer for 1 h. HeLa cells were cultured on confocal dishes (100350, SPL). Cells were transfected with HA-MYC- and RIPK3-GFP-expressing plasmids and then treated with the indicated chemicals. After treatments, the cells were fixed with 4% paraformaldehyde for 10 min at room temperature and then permeabilized via incubation with PBS containing 0.1% Triton-X 100. The samples were incubated with PBS containing 2.5% BSA and anti-HA antibody (1:100; sc-7392, Santa Cruz Biotechnology) for 2 h. The cells were washed with PBS three times and then incubated with Alexa Fluor 594 anti-mouse (1:200; A-11005, Thermo Fisher Scientific) for 1 h. DAPI was used to stain the nuclei. All stained samples were analyzed with a confocal microscope (LSM 800, Carl Zeiss).