Figure 3
The N-terminal region upstream of the LRRK2 leucine-rich repeat interacts with FADD. (a) A schematic of the domain structure of LRRK2 with amino acid boundaries used in this study to map the interacting domain with FADD (lower schematic). (b) V5-tagged FADD was co-expressed in HEK293T cells with Flag-tagged LRRK1, or Flag-tagged constructs of LRRK2, encoding the full-length protein, or each of the discrete domains as illustrated in (a). Cell lysates were incubated with anti-Flag resin and the inputs and eluates were separated by 12% SDS-PAGE, followed by probing the membranes with anti-V5, anti-Flag, or anti-β-actin. The upper blots show the immunoprecipitation of Flag-LRRK1 or Flag-LRRK2 and probed for anti-Flag, and the co-precipitation of V5-FADD is shown in the middle panel (IP eluate) probed with anti-V5. FADD interacts with full-length LRRK2, but not the related LRRK1, and binds only the N-terminal domain upstream of the LRR. The left blot (in panel [b]) shows a longer exposure of the region highlighted by the box. Identical amounts of protein from the input lysate from each sample was separated by 12% SDS-PAGE, and the membranes probed with anti-Flag, anti-V5, and anti β-actin (c). In the blot shown in (d), identical lysates from cells expressing Flag-LRRK1 or Flag-LRRK2 were separated by 8% SDS-PAGE, to allow better separation of the LRRK1 and LRRK2 proteins.
