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Figure EV5. Effects of PAWS1 on Wnt pathway components U2OS wild‐type (WT) and PAWS1−/− (KO) cells were treated with control‐conditioned medium or Wnt3A‐conditioned medium and lysed at the indicated time points. Extracts (0.5 mg protein) were subjected to IPs using anti‐CK1α or pre‐immune IgG control (10 μg antibodies coupled to 10 μl packed protein‐G sepharose beads). IPs were resolved by SDS–PAGE and immunoblotted with the indicated antibodies. Stable U2OS Flp‐In TRex cells were treated with 20 ng/ml doxycycline for 16 h, for induction of PAWS1‐GFP or GFP protein expression, and with Wnt3A conditioned medium or control medium for 3 h prior to lysis. GFP pull downs were resolved by SDS–PAGE and the gel was stained with Coomassie. Each lane was cut into five pieces, which were subsequently processed for protein identification by mass fingerprinting analysis. PAWS1‐GFP interacting proteins were plotted using total spectral counts for selected individual protein for both control (filled) and Wnt3A (open) conditions. Total spectral counts are defined as the sum of all the spectra associated with a specific protein within a sample, which includes also those spectra that are shared with other proteins. A spectral count of 3 or more in either control or Wnt3A condition in PAWS1‐GFP IPs and no spectral counts in GFP control IPs were set as threshold for inclusion. All proteins, except those indicated with asterisks, were identified as endogenous PAWS1GFP/GFP interactors as well (Fig 5A). PAWS1 phospho‐residues from gel slices 2 and 7 from (B) were identified by mass spectrometry. Residues denoted by “or” indicate that a specific single phospho‐residue on the corresponding tryptic peptide could not be assigned but could be any one of those indicated. U2OS wild‐type (WT) and PAWS1−/− cells were treated with control (L‐CM) or Wnt3A conditioned medium (L3‐CM) for 3 h and fractionated into cytoplasmic (C), nuclear (N), membrane (M) and cytoskeletal (Cs) fractions. Extracts (20 μg protein) from each fraction was resolved by SDS–PAGE, transferred onto PVDF membranes, which were probed by Western blotting with the indicated antibodies. α‐Tubulin was used as a cytosolic marker, Lamin A/C as a nuclear marker, LRP6 and EGFR were used as membrane markers, and Vimentin was used as a cytoskeletal marker. 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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