PC-9: 0,75 μg DNA + 1,5 μL jetPRIME in 96-well plate Lentivirus production

The selective down-regulation of activated intracellular proteins is a key challenge in cell biology. RHO small GTPases switch between a GDPbound and a GTP-bound state that drives downstream signaling. To date, no tool is available to study endogenous RHO induced conformational changes in live cells. Here, we set up an original cell-based screen to selectively degrade the RHOB-GTP fraction using nanobodies functionalized with a F-box domain. We identified one selective intrabody that shows selective targeting of endogenous RHOB-GTP mediated by interactions between the intrabody CDR3 loop and the GTP-binding pocket of RHOB. Our results indicate that, while RHOB expression is highly regulated, only the minor GTP-bound fraction but not its global expression mediates RHOB functions in genomic instability and in cell invasion. The F-box/intrabody-mediated protein degradation represents a unique approach to selectively target the active form of small GTPases or other proteins with multiple cellular activities.