Co-immunoprecipitation Analysis of GA50 and SQOR Interaction

We refer to the experimental procedures described in the previously published literature for co-immunoprecipitation analysis [38 (link)]. First, the pCMV/GA₅₀/Myc plasmid and pCMV/SQOR/HA plasmid were co-transfected into 293 T cells according to the transfection method described in Section 2.2. After 24 h, cell lysates were collected using Pierce™ IP Lysis Buffer (Thermo Fisher Scientific). Next, supernatants were immunoprecipitated with anti-Myc-Tag antibody or normal immunoglobulin G from rabbits for 2 h (4 °C). Beads of Protein G-Sepharose were then added for 1 h. Finally, the co-immunoprecipitation complexes were washed and identified by Western blotting using mouse anti-HA-Tag monoclonal antibody (Cell Signaling Technology). In addition, we performed immunoprecipitation of cell lysates with mouse anti-HA-Tag monoclonal antibody and Western blotting with rabbit anti-Myc-tag monoclonal antibody. In another set of experiments, we co-transfected pCMV/SQOR/Myc plasmids and pCMV/GA50/HA plasmids into 293T cells and performed co-immunoprecipitation as described above.