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Mass-spectrometric analyses For absolute quantification of ubiquitin linkages (Ub-AQUA/PRM), analytes were separated using short-run SDS-PAGE and the gel pieces were washed in 50 mM AMBC/30% acetonitrile (ACN) for 2 h, in 50 mM AMBC/50% ACN for 1 h, and dehydrated in 100% ACN for 15 min