Serine-38 of Ana2 is phosphorylated in vitro by Plk4 as identified by mass-spectrometry. from Two-step phosphorylation of Ana2 by Plk4 is required for the sequential loading of Ana2 and Sas6 to initiate procentriole formation

Supplementary Figure S2. Serine-38 of Ana2 is phosphorylated in vitro by Plk4 as identified by mass-spectrometry. Phospho-Mascot-interpreted fragmentation spectra of the peptide 13-LAPRP…EV (phospho-S-38) ILFG…SPR-65 with phosphorylated serine at the position 38 (highlighted red). Labelled peaks correspond to fragment ions. Despite the relatively long peptide, the fragmentation pattern unambiguously identifies the phosphorylation site

GST-Ana2-S38A can bind Sas6 after Plk4 phosphorylation from Two-step phosphorylation of Ana2 by Plk4 is required for the sequential loading of Ana2 and Sas6 to initiate procentriole formation

Supplementary Figure S3: GST-Ana2-S38A can bind Sas6 after Plk4 phosphorylation. Sas6 specifically interacts with both Ana2-WT and Ana2-S38A when the proteins are pre-phosphorylated by Plk4. GST, GST-tagged wild-type (GST-Ana2-WT) or the S38A substitution mutant (GST-Ana2-S38A) were treated with either MBP-Plk4 or MBP-Plk4KD and incubated in vitro with 35S-Met-labeled Sas6 produced in a coupled in vitro transcription/translation reaction. The resulting complex was analyzed by SDS-PAGE and autoradiography

List of site-directed mutagenesis primers from Two-step phosphorylation of Ana2 by Plk4 is required for the sequential loading of Ana2 and Sas6 to initiate procentriole formation

List of site-directed mutagenesis primer

Plk4 phosphorylation induces a band-shift in Ana2 from Two-step phosphorylation of Ana2 by Plk4 is required for the sequential loading of Ana2 and Sas6 to initiate procentriole formation

Supplementary Figure S1. Plk4 phosphorylation induces a shift in the electrophoretic mobility of Ana2. A. Incubation of Ana2 synthesized by coupled in vitro transcription and translation (IVTT) with active MBP-Plk4 in the presence of increasing ATP induces a distinct band-shift. B. The shift in mobility of Ana2-FLAG protein following its co-overexpression with active non-degradable Plk4 (Plk4ND) is abolished by treating the extract with λ-Phosphatase. C. Alanine substitutions in mass-spectrometry-identified phospho-sites with highest spectral count following in vitro phosphorylation, including those of the STAN motif, still retain the band-shift. D: Phosphorylation sites identified in Ana2 by mass-spectrometry. Serine or threonine residues identified following phosphorylation by MBP-Plk4 in vitro are highlighted in blue. Sites identified as phosphorylated in vivo on tagged-Ana2 (Protein A, FLAG, or GFP tags) purified from D.Mel-2 cells or early Drosophila embryos are highlighted in yellow. The STAN-motif is highlighted in grey. E. The phosphorylation-site responsible for the band shift is located in the N-terminal 280 amino acids of Ana2. Ana21-280, but not Ana2281-420 displays the band-shift in both the co-overexpression assay (upper) and the in vitro phosphorylation assay (lower). F. Band-shift assay using IVTT product of WT and non-phosphorylatable mutants of Ana2 for the sites highlighted in yellow within the N-terminal 280 amino-acid part. The band-shift is seen for each of these mutants

Additional file 1: Figure S1. of DAPPER: a data-mining resource for protein-protein interactions

An overview of DAPPER retrieval tools. Venn diagram highlighting sections of records which will be retrieved by various tools, when applied to three hypothetical experiments. Each of the three tools can retrieve data in either tab-delimited or HTML format (see DAPPER Results web page for these options). Figure S2. Home page of DAPPER. The search field can be used to retrieve all experiments containing a particular CGID of Drosophila melanogaster. Browse button (MartView link) navigates to DAPPER BioMart interface for custom querying. By default, results are sorted (descending) by “Protein score”. Upload link is currently restricted to DAPPER curators and collaborators only. Figure S3. Stepwise guide to retrieve all proteins and their ontological annotations for a given experiment. The guide also shows how data from an external resource (Ensembl) can be retrieved and seamlessly integrated with DAPPER contents. Figure S4. Stepwise guide to retrieve kinetochore associated Drosophila genes. Gene ontology filter (GO Term Name) is set to “kinetochore” through linking with Ensembl database. Entries retrieved are further sorted by “Protein score” using SORT tool (SORT_HTML) on the Results page. Figure S5. Stepwise guide to compare two (or more) experiments. The results will contain only those hits which are common between the experiments selected for comparison (BubR1 and Polo kinase). Experiments having an entry in common are grouped together for better interpretation of results. (PDF 2334 kb

Additional file 2: Table S1. of DAPPER: a data-mining resource for protein-protein interactions

List of unique Baits in DAPPER. (XLSX 31 kb

Expression levels in Ana2-Myc cell lines from Two-step phosphorylation of Ana2 by Plk4 is required for the sequential loading of Ana2 and Sas6 to initiate procentriole formation

Supplementary Figure S4: Expression levels in Ana2-Myc cell lines: An immuno-blot showing comparable levels of expression between an Ana2-WT-Myc and an Ana2-S38A-Myc cell line. The same three cell lines are presented as in Fig. 6B: Untransfected D.Mel-2 cells (lane 1), pAct5-Ana2-WT-Myc (lane 2) and pAct5-Ana2-S38A-Myc (lane 3). Top panel: anti-Asl immuno-blot as a loading control. Bottom panel: anti-Ana2 immuno-blot, revealing endogenous Ana2 and overexpressed Ana2-Myc