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. 2011 Jan 19;31(3):907–912. doi: 10.1523/JNEUROSCI.5092-10.2011

Figure 1.

Figure 1.

Comparable expression and oligomerization of WT and mutant G2019S LRRK2. A, Western blot analysis of FLAG-LRRK2 expression. HEK 293T cells were infected with an equal amount of viral particles of rAd-WT-LRRK2 or rAd-G2019S-LRRK2 (n = 3 per group). A noninfected (NI) control was included. rAd-WT-LRRK2- and rAd-G2019S-LRRK2-injected animals were killed at 10 d after injection (n = 4 per group). Protein extracts from each sample were subjected to SDS gel electrophoresis and membranes were probed with anti-FLAG and anti-actin antibodies. Both vectors were found to drive equivalent levels of expression of FLAG-LRRK2 in vitro and in vivo, following densitometric analysis and normalization to actin levels. B, Fold human LRRK2 overexpression in the SN. rAd-WT-LRRK2- and rAd-G2019S-LRRK2-injected animals were killed at 14 d after injection (n = 4 per group). Protein extracts from uninjected (−) and injected (+) SN were separated by SDS gel electrophoresis. Membranes were probed with an anti-LRRK2 antibody that recognizes rodent and human LRRK2. The fold overexpression of human LRRK2 in each individual rat was estimated by comparing LRRK2 signal intensities, normalized to actin, between the uninjected and injected SN. Further probing of the membrane with anti-FLAG antibody confirmed the rAd-driven expression of FLAG-LRRK2 in all injected SN. An average twofold overexpression of human LRRK2 was measured for both the WT and G2019S groups. C, Native PAGE analysis of endogenous and overexpressed human LRRK2. A fraction of the soluble protein extracts of dissected striata and SN were analyzed by native PAGE. Upon staining with an anti-LRRK2 antibody, endogenous LRRK2 in the striatum and SN was shown to assemble into high-molecular-weight complexes ranging from 480 kDa to >1 MDa. Despite loading up to 100 μg of total proteins, we could not detect any protein migrating at the size expected for monomeric LRRK2 neither in the SN, nor in striatal extracts. Overexpressed WT and G2019S LRRK2, detected by anti-FLAG immunostaining, showed a qualitatively similar higher-order distribution compared to each other and endogenous LRRK2. Uninjected striatal and nigral extracts were loaded as controls for the specificity of the anti-FLAG signal. Despite the presence of a nonspecific band at ∼720 kDa upon FLAG staining on SN lysates, we could clearly detect higher-order complexes between 480 and 720 kDa for overexpressed LRRK2 in the SN.