BOR-Syndrome-Associated Eya1 Mutations Lead to Enhanced Proteasomal Degradation of Eya1 Protein
Figure 2
Six proteins stabilize Eya1 and Eya1 mutants with the exception of L550P.
(A) COS-7 cells were transfected with empty vector (control), HA-Eya1 or mutants, and FLAG-Six2. Equal amounts of protein were analyzed by SDS-PAGE and immunoblotting using anti-HA and anti-FLAG antibody. (B) Endogenous Eya1 is stabilized in presence of Six2. MK4 cells were transfected with Eya1-specific or non-target control siRNA and Eya1 protein levels were detected by immunoblotting using anti-Eya1 antibody (left). MK4 cells stably expressing FLAG-Six2 were subject to immunoblot analyses using Eya1-specific antibody (right). Asterisk indicates unspecific signal. (C) Six induces accumulation of Eya1 protein in different cell lines via the conserved Six and homeo domain. COS-7 and U2OS cells were transfected with HA-Eya1 and expression vectors for Six1, Six2, C-terminal domain of Six2 (CD), GDNF and H-Ras. Eya1 protein levels were detected by immunoblotting using anti-HA antibody. (D) Schematic representation of the Six2 deletion constructs used for characterization of Eya1 stabilization (left). COS-7 cells were co-transfected with HA-Eya1, Six2 or deletion constructs as indicated. Eya1 protein levels were analyzed by immunoblotting.