Primary antibody against yap

CRC cells were seeded on chamber slides until adhered and cocultured with F. nucleatum (MOI of 100:1) for 2 h. After washing with PBS, the cells were fixed with 4% paraformaldehyde for 15 min at room temperature (RT). The slides were incubated with 0.2% Triton X-100 for 15 min and blocked in 3% BSA for 30 min. After washing by PBST three times, the cells were incubated with primary antibody against YAP (Proteintech, China) at RT for 2 h. Then the slides were washed by PBST three times and incubated with secondary antibodies and DAPI at RT for 1 h. The cells were fixed with Gold Antifade Reagent (Invitrogen, USA) and imaged using LSM 800 with Airyscan confocal laser-scanning microscope (Zeiss, Germany).

Cells were seeded onto glass coverslips placed in 24-well plates and cultured overnight at 37 °C. Then, the cells were fixed with 4% formaldehyde for 15 min and permeabilized with 0.1% Triton X-100 in PBS for 15 min at room temperature. After washing three times with PBS, the cells were blocked with 1% bovine serum albumin for 30 min and incubated with primary antibody against YAP (proteintech, 1: 250) overnight at 4 °C. Subsequently, the cells were incubated with an Alexa Fluor® 550-conjugated secondary antibody (Jackson Immuno Research, 1:1000) for 30 min and stained with DAPI (Sigma-Aldrich) for 5 min in the dark at room temperature. Finally, the cells were observed and photographed under a fluorescence microscope (Olympus, Tokyo, Japan).