Effect of human mutant-type FRMD7 proteins on the activation of Rac1 signaling.

<p>HA-tagged human Rac1 was co-transfected into HEK293T cells with Flag-tagged wild-type (Wild) or mutant-type (c.781C>G, c.886G>C and c.1003C>T) human FRMD7, cultured for 48 hours, and lysed. Cell lysates were clarified by centrifugation, and the supernatant was incubated with GST-PAK2 protein immobilized on glutathione-sepharose beads, where bound GTP-Rac1 proteins were detected by Western blotting with anti-HA monoclonal antibody. Extracts of HEK293T cells transfected with two missense mutant-type FRMD7 (c.781C>G, C781, c.886G>C, G886) showed decreased amounts of GTP-Rac1 compared with the wild-type FRMD7. Little activation of Rac1 was detected in extracts of HEK293T cells transfected with another truncated mutant-type FRMD7 (c.1003C>T, C1003). PC3.1: pcDNA3.1+Rac1; CON: Rac1 only. The experiments were repeated five times, where the graphs represent the average of five independent experiments (columns, mean; bars, S.E.M.; *p < 0.05, **p < 0.01).