PARK2 Modifies Synucleinopathy Figure 3. The solubility of PS129-a-synuclein but not PS87-a-synuclein species is decreased in the brains of end-stage symptomatic hA30Pa-syn mice. (A) Representative western blot using protein-specific antibodies illustrating recognition of human a-synuclein and its isoforms phosphorylated at serine 87 or 129, in the TBS-soluble brain fraction of a symptomatic transgenic hA30Pa-syn mouse. These proteins were not detected in the TBS- or SDS-soluble brain fractions of an aged wild-type control animal (left panel). The anti-PS87- and anti-PS129-a-synuclein antibodies detected specifically normal recombinant a-synuclein (PS87,129a-syn), phosphorylated in vitro by casein kinase 1 as described previously [42], but not the non phosphorylated protein (a-syn). As expected, the anti-PS87-a-synuclein antibody also recognized the in vitro-phosphorylated S129A a-synuclein variant (S129A PS87a-syn), but not phosphorylated S87A a-synuclein (S87A PS129a-syn). In contrast, the anti-PS129-a-synuclein antibody recognized S87A PS129a- syn but not S129A PS87a-syn. (B) Representative western blots illustrating a-synuclein (top panel), PS87-a-synuclein and PS129-a-synuclein (middle and bottom panels) protein abundance in TBS-soluble and -insoluble (SDS) brain fractions from symptomatic (S) and non symptomatic (ns) mice with and without Parkin. (C) Quantitative analysis of human a-synuclein, PS87-a-synuclein and PS129-a-synuclein solubility in symptomatic and non symptomatic hA30Pa-syn mice with (ns: n = 4; S: n = 1328) and without (ns: n = 4; S: n = 1128) Parkin. The relative protein amount was defined as the ratio between the amount of protein normalized to a-tubulin in the SDS fraction, and the total amount of protein normalized to -tubulin in the SDS and TBS fractions. The abundance of PS129-a-synuclein in the SDS brain fraction was significantly higher in symptomatic mice in the presence or absence of Parkin than in healthy mice. *, p,0.01. (D) Representative western blots showing PS129-a-synuclein immunoreactive species with retarded electrophoretic mobility after long exposure times in the SDS fraction from symptomatic mice. (E) Quantitative analysis of the monomer (short exposure times) and the PS129-a- synuclein species with higher apparent molecular mass (long exposure times) in symptomatic mice with (n = 13) and without (n = 11) Parkin. (F) Western blot analysis with anti-Parkin antibodies of TBS and SDS fractions from a wild-type mouse, a representative pool of healthy and symptomatic hA30Pa-syn mice, and a symptomatic Parkin-deficient hA30Pa-syn control mouse. Parkin was abundant in the TBS fractions but was not detected in the SDS fractions from healthy or symptomatic mice, even after long exposure times. The asterisk indicates a non specific band recognized by anti-Parkin antibodies. doi:10.1371/journal.pone.0006629.g003 PLoS ONE | www.plosone.org 4 August 2009 | Volume 4 | Issue 8 | e6629