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Figure EV2. PAWS1 is phosphorylated at Ser614 by CK1α in vitro 32P autoradiography and Coomassie stain of SDS–PAGE gel after an in vitro kinase assay with GST‐CK1α and GST‐PAWS1‐6xHis as a substrate.GST‐PAWS1‐6xHis phosphorylated by CK1α in A was excised from the gel, digested with trypsin and resolved by HPLC on a C18 column using increasing acetonitrile gradient. Analysis of the [γ32P] radioactivity peak at 54.1 min (P1) by LC‐MS/MS revealed the phospho‐peptide RPSVASSVSEEYFEVR.Analysis of the [γ32P] radioactivity peak P1 by LC‐MS/MS revealed various phospho‐peptides, of which RPSVASS(P)VSEEYFEVR was the only one to match the solid‐phase Edman sequencing data. Together, they reveal that CK1α phosphorylates PAWS1 at Ser614. 32P autoradiography and Coomassie stain of SDS–PAGE after an in vitro kinase assay with CK1αWT or CK1αKD (kinase dead) and PAWS1WT or PAWS1S614A as substrates.Human PAWS1WT, hPAWS1S610A and hPAWS1S610A/S614A induce axis duplication in Xenopus embryos. Image published in: Bozatzi P et al. (2018) © 2018 The Authors. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license Larger Image Printer Friendly View

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