Phosphorylation-Dependent 14-3-3 Binding to LRRK2 Is Impaired by Common Mutations of Familial Parkinson's Disease
Figure 2
Identification of 14-3-3s as LRRK2 interaction proteins.
(A) Coomassie blue staining showing the affinity-purified FLAG-LRRK2 and its binding proteins from FLAG-LRRK2 transgenic brain. The positions of the FLAG-LRRK2 and 14-3-3 bands are labeled with arrows. Wild-type mouse brain was used as the control for background proteins during purification. (B) Western blot analysis was performed to detect the presence of various 14-3-3 isoforms in association with the affinity purified FLAG-LRRK2 protein. Antibodies against specific isoforms of 14-3-3 were used. Wild type mouse tissue was used as the control. (C) FLAG-LRRK2 and different Myc-14-3-3 isoform plasmids were co-transfected into HEK-293T cells and Immunoprecipitation was performed using anti-Myc antibody, followed by the analysis of protein levels using indicated antibodies. (D) FLAG-LRRK2 plasmid was transfected into HEK-293T cells and transfected cell lysate was incubated with different purified GST-14-3-3 isoforms as indicated. The GST-pull down assay was assayed by Western blot analysis of the FLAG-LRRK2.