Ripk2

Immunofluorescence analysis was performed as described previously (Albert et al, 2020 (link)). In brief, HCT116 or HeLa cells were seeded on coverslips in 12‐well plate and transfected with GFP‐XIAP for 16 h. Cells were washed with PBS and subsequently fixed with 3% paraformaldehyde in PBS for 20 min. Cells were blocked and permeabilised with blocking buffer (0.1% saponin (Carl Roth), 3% BSA (Carl Roth) in PBS) for 30 min and later incubated with primary antibodies for TAB1 (1:100, Cell Signaling) or RIPK2 (1:100, Cell Signaling) in a humid chamber overnight at 4°C. After incubation, coverslips were washed with washing buffer (0.1% saponin in PBS) three times and incubated with secondary antibody goat anti‐rabbit Alexa Fluor 568 (1:500, Thermo Fisher Scientific) for 1 h at room temperature. Subsequently, cells were stained with 300 nM DAPI (Molecular Probes) for 10 min and washed three times and embedded with mowiol overnight. For imaging, Fluoview FV1000 confocal microscope (Olympus GmbH) was used (objective: Olympus PlanApo, 60×/1.40 oil, ∞/0.17).