Figure 2.

Phosphorylation of α-synuclein in MsrA ^−/− and wild-type (WT) brain extracts. (a) Tris-soluble and Urea-soluble brain extracts (40 μg protein) of both mouse types were prepared as described in Section 2. These extracts were then incubated in the presence of additional brain-matched Tris-soluble extract (10 μg protein, serving as a source for kinases), 25 mM Tris (pH 7.4), protease inhibitor cocktail (no-EDTA) (Roche), 1 mM CaCl₂, 10 mM MgCl₂ and 16.7 μM [γ-³²P]-ATP for 3 minutes at room temperature in a final volume of 50 μL. Endogenous phosphorylation was stopped by addition of 10 mM EDTA, 10 mM EGTA, and 1 mM cold ATP and immediately placed on ice. Then, the samples were subjected to an Immunoprecipitation by anti-α-synuclein antibodies or anti-MetO antibodies as described in Section 2. Thereafter, equal protein amounts of the immunoprecipitants were subjected to an SDS-gel electrophoresis (4%–20%) followed by exposure of the gel to an X-ray film. Lanes 1, 3, 5, and 7 represent Tris-soluble fractions and lanes 2, 4, 6, and 8 represent Urea-soluble fractions. Syn, α-synuclein; ab, antibodies; kDA, molecular mass markers in kilo-Dalton. The detected band following the immunoprecipitation by anti-MetO antibodies was also denoted in the text as MetO-15. (b) Densitometry analysis of authoradiographed bands detected in (a). The percent MetO values were calculated as percent ratio of the detected immunoprecipitant [γ-³²P]-α-synuclein in the Urea-soluble fractions, in which WT's [γ-³²P]-α-synuclein-averaged value represents 100%. The statistical difference between the [γ-³²P]-α-synuclein levels in both mouse types was significant (n = 5; *P < .05 by t-test). (c) Densitometry analysis of the phosphorylated α-synuclein bands detected in Western blot analysis (a representative blot is shown in a small window) after isolation of phosphorylated proteins from WT and MsrA ^−/− brains (using PhosphoProtein Purification Kit (Qiagen)). See Section 2 for relevant procedures and analyses. The statistical difference between the phosphorylated α-synuclein levels in both mouse types was significant (n = 5; *P < .05 by t-test).