Fig. 3.

Nbs bind LRRK2 via interactions with different domains. (A) Mapping of the Nb-binding epitopes using CL-MS. The Nbs are divided into five groups according to their effect on LRRK2 kinase activity, as defined in Fig. 1B. The observed cross-links between the Nbs and LRRK2 are indicated by lines, with the corresponding lysine residues on LRRK2 indicated by their residue number. The domain specificity of the Nbs, as determined in ELISA, is given below the respective Nbs. (B) Nbs immunoprecipitate endogenous (mouse) LRRK2. Lysates derived from RAW264.7 cells were incubated with 1.5 µM purified His-tagged Nbs or an irrelevant control Nb (Irr Nb), and pull-downs were performed using magnetic Dynabeads. As a positive control (+ cntrl) LRRK2 was pulled-down using a LRRK2-specific Nb. LRRK2 was detected via immunoblotting (IB) using two different antibodies (C-412 and 24D8). The blot is representative of n = 3.