To validate this suppression of Hh signaling by Dyrk2-deletion, we performed a transient over-expression experiment using wild-type human DYRK2 or a DYRK2-K251R construct that expresses a kinase dead mutant (Taira et al., 2012; Figure 3—figure supplement 1C–D) in Dyrk2-/- MEFs using adenovirus infection (Yokoyama-Mashima et al., 2019). Here, DYRK2 is linked to adenoviridae infectious disease.