Fig. 3

Experimental design. a: FLAG-DVL3 was overexpressed (with or without kinase) in HEK293 cells. After cell lysis DVL3 was immunoprecipitated using anti-FLAG antibody. Immunoprecipitates were separated on SDS-PAGE gel electrophoresis, stained with Coomassie brilliant blue and the 1D bands corresponding to DVL3 were excised, digested with trypsin and subsequently cleaved by chymotrypsin. In the pipeline #1, the aliquot (1/10) of concentrated sample was directly analyzed by LC-MS/MS in order to analyze site occupancy of the abundant phosphorylated sites. The rest of the sample was enriched for phosphorylated peptides using TiO₂ and analyzed by LC-MS/MS to obtain detailed information of DVL3 phosphorylation status. Data from LC-MS/MS were searched, manually validated in Skyline software and further processed by two approaches. In the first approach (pipeline #2) only phosphorylated peptides with the clearly localized phosphorylated site (based on manual inspection of spectra) were considered. In the second approach (pipeline #3) we have considered all phosphorylated peptides that in some cases resulted in the formation of “clusters” of phosphorylated sites. b: The overall sequence coverage of DVL3 across all kinases and replicates. Regions of DVL3 covered by the peptides detected (Mascot score > 20) in any of the MS/MS analyses are highlighted in grey. For sequence coverage in individual samples see Additional file 6: Table S2