Insights into gli factors ubiquitylation methods

The Hedgehog (Hh) signaling pathway governs cell growth and tissue development. Malfunctioning of several Hh pathway components, including the key transcriptional effector Gli proteins, is responsible for the onset of several tumors. Gli proteins activity is finely controlled by multilayered regulatory mechanisms, the most prominent of which is their proteasome-dependent proteolytic cleavage or massive ubiquitin-mediated proteolysis. Here, we described multiple procedures to determine whether a Gli protein is ubiquitylated both in a cellular context and in vitro, in basal conditions or by different E3 ubiquitin ligases and whether these processes are associated to Gli proteasome degradation.