Considerations in the Identification of Endogenous Substrates for Protein L-Isoaspartyl Methyltransferase: The Case of Synuclein
Figure 5
[3H] methyl blot and Western blot analysis of an α/β-synuclein immunoprecipitation from PIMT wild type and knockout mouse brain extracts.
Equal amounts (400 µg) of protein from each genotype were immunoprecipitated with a mouse monoclonal antibody specific to α/β-synuclein. Brain extracts (20 µg per lane; lanes 1 and 2), and immunoprecipitates (lanes 3–6) were separated by 1D SDS-PAGE before electroblotting onto PVDF. The membrane was then subjected to [3H] methyl blot analysis as in Figs. 3 and 4, followed by a 48 h exposure to a tritium-sensitive phosphor imager screen. Using the same membrane, a Western blot (right panel) was performed with rabbit anti-pan synuclein (1∶10,000). A strong immunoreaction to α/β-synuclein was seen in lanes 4 and 6, but not in lanes 3 and 5 where the anti-synuclein antibody was omitted from the immunoprecipitation. The positions of the IgG heavy chain (HC), IgG light chain (LC), and synuclein (syn) are indicated on the far right edge. The dark bands at ∼45 kDa and ∼120 kDa in lanes 1 and 2 of the Western blot are due to proteins in the brain extract that cross reacts with anti-synuclein, similar to what is seen in panels B and D of Fig. 4.