Figure EV2. PAWS1 is phosphorylated at Ser⁶¹⁴ by CK1α in vitro .

1. 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.
2. 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.
3. 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.
4. ³²P autoradiography and Coomassie stain of SDS–PAGE after an in vitro kinase assay with CK1α^WT or CK1α^KD (kinase dead) and PAWS1^WT or PAWS1^S614A as substrates.
5. Human PAWS1^WT, hPAWS1^S610A and hPAWS1^S610A/S614A induce axis duplication in Xenopus embryos.