Paleologou KE, Oueslati A, Shakked G, Rospigliosi CC, Kim HY, Lamberto GR, Fernandez CO, Schmid A, Chegini F, Gai WP, Chiappe D, Moniatte M, Schneider BL, Aebischer P, Eliezer D, Zweckstetter M, Masliah E, Lashuel HA. Phosphorylation at S87 is enhanced in synucleinopathies, inhibits alpha-synuclein oligomerization, and influences synuclein-membrane interactions. J Neurosci. 2010 Mar 3;30(9):3184-98. PubMed Abstract

REAGENTS/MATERIAL:
Immunocytochemistry, S87-P in transgenic mice: To investigate the distribution of the phosphorylated α-syn epitopes brains from non-TG and TG mice, seriallysectioned, free-floating, blind-coded vibratome sections were incubated overnight at 4°C with either the mouse monoclonal anti-α-syn (42) (Transduction Laboratories), rabbit anti-human α-syn (Millipore Bioscience Research Reagents), mouse monoclonal anti-S129-P α-syn (pSyn 64) (Waco Chemical Industries) and the rabbit polyclonal anti-S87-P α-syn, as described previously (Masliah et al., 2000).
Analysis of injected rat brains, Immunofluorescence: Slices were incubated overnight at 4°C with rabbit anti-tyrosine hydroxylase (Millipore) and mouse monoclonal anti-Human α-syn (LB509) (Zymed Laboratories) antibodies in blocking buffer and subsequently incubated for 2 h at room temperature with secondary antibodies conjugated to Alexa Fluor-488 or Alexa Fluor-568. Slices were incubated overnight at 4°C with the primary antibody anti-P Ser 87.
Double labeling and confocal microscopy: To determine the colocalization between α-syn-immunolabeled neurons and CK1, 40-μm-thick vibratome sections from α-syn TG mice were immunolabeled with the rabbit polyclonal anti-α-syn (Millipore Bioscience Research Reagents) and polyclonal anti-CK1δ (C-18) (Santa Cruz Biotechnology).
Detection of S87-P in Lewy bodies: Lewy body enrichment was conducted as previously described (Gai et al., 2000). Primary antibodies were affinity-purified sheep anti-α-syn (Gai et al., 1999), affinity purified rabbit anti-S87-P α-syn, and mouse monoclonal anti-pS129 α-syn (11A5) (Anderson et al., 2006).
In vitro phosphorylation and dephosphorylation assays: The progress of the phosphorylation reaction was monitored by Western blot using antibodies against S87-P and S129-P.
Gel electrophoresis (SDS-PAGE) and immunoblotting: α-Syn samples were diluted in loading buffer and separated on 12% SDS 1 mm gel. Gels were stained with Simply Blue Safe stain (Invitrogen) or silver stained (Invitrogen) according to manufacturer’s instructions. For Western blots, membranes were probed with mouse monoclonal anti-α-syn (121–125) (211) (Santa Cruz Biotechnology) or mouse monoclonal anti-α-syn (15–123) (42) (BD Transduction), or mouse monoclonal anti-S129-P α-syn (pSyn64) (Wako), or rabbit polyclonal anti-S87-P α-syn. Western blot analysis for brain homogenates used the following primary antibodies: rabbit polyclonal anti-total α-syn (Millipore Bioscience Research Reagents), mouse monoclonal anti-S129-P α-syn (courtesy of Dr. T. Iwatsubo, University of Tokyo, Tokyo, Japan), rabbit polyclonal anti-S87-P α-syn and mouse monoclonal anti-α-actin (ASM-1) (Millipore Bioscience Research Reagents).