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Figure S1. �2 isphosphorylatedatThr-156.DialysedAP2 (from the hydroxyapatite derivedpool 4, 200�g) wasphosphorylatedin4 ml with 40 �l [{gamma}32P]-ATP (3000 ci/mmol, Amersham).After 15 minatRTa secondincubationfor 15minwasperformedby adding unlabeledATP (final concentration2mM).The reactionwasstoppedby adding 400 �l 100% TCA onice.After 10h incubationthe sample wascentrifugedat14000rpm andthe derivedpelletwasresuspendedinsample buffer, resolvedby SDS-PAGE andvisualizedby Coomassie staining.The 50kDa bandwasexcised, cutin1mm small piecesandsubjectedto tryptic digestion (substrate-enzyme ratio 100:3) for 12 h.The mixture containing the tryptic peptideswassubsequently filtered(0.2 �m pore diameter, Costar) andappliedto reverse-phase chromatography using C18 (Pharmacia).The peptideswere elutedwith a linear gradientof 0-100% acetonitrile in0.1% trifluoroacetic acid(TFA) within60 minata flow rate of 20�l/minduring which automatic peak fractionationwasperformed.The radioactivity ineach fractionwasmonitoredby Cerencov counting.Aliquotscontaining radioactive peptideswere analyzedby massspectrometry (MALDI/MALDI MS) or immobilizedona PVDF matrix for phosphoamino acidsequencing with a Sequelon-AA ReagentKit(Millipore).Two peptideswere detectable by MALDI/MALDI MS: one comprising the AP2 �-subunitresiduesGlu146-Arg162 anda longer peptide corresponding to the �2 residuesSer140-Arg162.The presence of the latter peptide isdue to the incomplete trypsindigestionatpositionLys145.The massof the indicatedpeptidescorrespondedto the single phosphorylatedform.Radio sequencing of the peptidespresentinfraction19 and20 revealedthatthey were phosphorylatedata single positioncorresponding to residue Thr-156.