Figure 3. Novel mutation of FRMD7 downregulates the activation of Rac1 signaling HA-tagged human Rac1 was co-transfected into HEK293T cells with Flag-tagged wild-type (Wild) or mutant-type (c.635T>C) FRMD7. The supernatant of cell lysates was incubated with GST-PAK2 protein immobilized on glutathione-sepharose beads, where bound GTP-Rac1 proteins were detected by Western blotting using anti-HA monoclonal antibody. The amount of input HA-Rac1 and Flag-FRMD7 detected by anti-HA or anti-Flag monoclonal antibody. Extracts of HEK293T cells transfected with wild-type FRMD7 could detect the PAK2 precipitation GTP-Rac1 band, however mutant-type FRMD7 contained decreased amounts of GTP-Rac1 compared with the wild-type (A). (Wild: wild-type FRMD7+Rac1; C635: mutant-type (c.635T>C) FRMD7+Rac1; Control: empty vector+Rac1). The experiments were repeated five times, and the graphs represent the average of five independent experiments (B) (Columns, mean; bars, SEM; *p<0.05).