Optimizing appropriate dissection site of firefly luciferase for BiLC Rho GTPase biosensor.

<p>(A) The optical imaging results among different split-sites of firefly luciferase. Relative luciferase activities were detected in living 293 cells cotransfected with the five different combinations of split firefly luciferase fragments (Nfluc416-WASP/Cfluc398-CDC42, Nfluc416-WASP/Cfluc417-CDC42, Nfluc437-WASP/Cfluc438-CDC42, Nfluc398-WASP/Cfluc384-CDC42, Nfluc445-WASP/Cfluc446-CDC42), respectively. In each luciferase fragments combination, luciferase activity was compared among CDC42 WT, G12V, and F37A mutants. WT, G12V, and F37A indicate wild type, the constitutively active mutant, and the effector-loop mutant, respectively. The results were normalized using cotransfection of renilla luciferase plasmid (pRL-tk) and represented by the ratio of luminescent intensity of firefly luciferase (FL) at 600 nm to that of renilla luciferase (RL) at 500 nm (FL/RL). The data shown are representative of four separate experiments performed with quadruplicate culture wells. The result shows that the combination (Nfluc416/Cfluc398) had the widest dynamic range and the highest luciferase activity restoration. (B) The western blots carried out in parallel to demonstrate the protein expression among CDC42 WT, G12V, and F37A biosensors. This figure only shows the results of Nfluc416-WASP/CDC42-Cfluc398 as representative.