Anti flag anti ha

To detect the interaction of endogenous TRIM69 with PRKCD, AGS, MKN28 or MKN45 cell lysates were obtained by using IP lysis buffer (Beyotime, Shanghai, China) and incubated with 2 µg of an anti-PRKCD antibody or anti-rabbit IgG (Beyotime, Shanghai, China) overnight at 4°C. Protein A + G magnetic beads (Beyotime, Shanghai, China) were added to the cell mixtures and incubated for 4 h at 4°C. For the interaction between exogenous TRIM69 and PRKCD, AGS, MKN28, MKN45 or HEK293T cells were transfected with TRIM69 or PRKCD plasmids. After culture for 48 h, the cells were homogenized in lysis buffer for immunoprecipitation (Beyotime, Shanghai, China). Then, the cell lysates were incubated with 20 µl of anti-IgG magnetic beads (Beyotime, Shanghai, China) overnight at 4°C. The next day, the mixtures were incubated with anti-Flag, anti-HA or anti-Myc magnetic beads (Beyotime, Shanghai, China) for 4 h at 4°C. After the beads were washed four times with lysis buffer for IP, the precipitates were collected, and proteins were eluted with 1×SDS sample buffer. The samples were analyzed using western blotting.