Fig. 1

Tissue processing and reverse phase arrays (RPAs). A Workflow for tissue processing towards biochemical analysis of brain tissue extracts. For detailed description, see “Methods”. B Schematic of aSyn with number of antibodies per epitope group included in RPA analysis indicated above the schematic (see also Additional File 1: Table S2). 23E8 and Syn-1 antibodies were also used in alphaLISA® to measure ‘Total aSyn’. Arrow heads indicate epitopes for antibodies specific and selective against posttranslational modification 119CTT, 122CTT, and pSer129 and some were used together with 23E8 in alphaLISA® for the quantification of the respective forms of aSyn. C Workflow for RPAs: samples were spotted on nitrocellulose film slides. Pairs of pads were first incubated with each to generate two replicate incubations for all included antibodies, and afterwards with the species-matching fluorescently labeled secondary antibody. For detailed description, see “Methods”. D Example of two scanned slides containing duplicates for 14 antibodies in the analysis. Abbreviations: RIPA: radio-immunoprecipitation assay buffer containing protease and phosphatase inhibitors; UTC, extraction buffer containing urea, thiourea and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate (CHAPS); SN, substantia nigra