Journal: Proceedings of the National Academy of Sciences of the United States of America
Article Title: Maternal Wnt/STOP signaling promotes cell division during early Xenopus embryogenesis
doi: 10.1073/pnas.1423533112
Figure Lengend Snippet: Gsk3 negatively regulates mitotic effectors in Xenopus extracts. (A) Scheme of the protein microarray experiment to assess Gsk3-dependent polyubiquitination. Interphase egg extracts were supplemented with ubiquitin, pretreated with LiCl or NaCl (Control), and then applied on protein arrays containing ∼9,000 proteins. Polyubiquitination of arrayed proteins (PolyUb) was monitored using anti-polyubiquitin antibodies and immunofluorescence detection using an array scanner. Signal intensities were transformed and are displayed as heat-map. (B) Distribution plot of polyubiquitination changes of 3,873 in vitro polyubiquitinated proteins after Gsk3 inhibition by LiCl (Log2[Control/LiCl]). Arrays were treated with 30 mM LiCl or 30 mM NaCl (Control). LiCl reduced polyubiquitination of 864 proteins >1.8-fold. Some candidates, associated with cell cycle progression and selected for further validation are indicated in the plot. (C) Cluster of mitotic associated proteins that are significantly regulated by LiCl in B. The functional network was determined using String 9.1 and color-coded based on the fold change in the microarray (Dataset S1). (D) Western blots of indicated proteins from Xenopus eggs (naturally arrested in metaphase II) and released into interphase with the Ca2+ ionophore A23187 for 25 min, treated with LiCl, or mock treated (NaCl). β-cat, β-catenin; cycE, cyclin E; Interph., interphase; M. II, metaphase II; pH3, phosphorylated histone 3. (E) Western blots of stage VI oocytes in Prophase I and Progesterone-matured (Prog.) oocytes in metaphase II. Oocytes were injected (Inj.) with Morpholinos (MO) or mRNA as indicated. Co, control; dnWnt11, dominant negative Wnt11; pH3, phosphorylated histone 3; PPL, preprolactin (negative control).
Article Snippet: Briefly, the arrays were washed in TBST and blocked for 4 h at 4 °C using Microarray Blocking solution (Arrayit), followed by a wash in lysis buffer.
Techniques: Microarray, Ubiquitin Proteomics, Control, Immunofluorescence, Transformation Assay, In Vitro, Inhibition, Biomarker Discovery, Functional Assay, Western Blot, Injection, Dominant Negative Mutation, Negative Control
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![Gsk3 negatively regulates mitotic effectors in Xenopus extracts. (A) Scheme of the protein <t>microarray</t> experiment to assess Gsk3-dependent polyubiquitination. Interphase egg extracts were supplemented with ubiquitin, pretreated with LiCl or NaCl (Control), and then applied on protein arrays containing ∼9,000 proteins. Polyubiquitination of arrayed proteins (PolyUb) was monitored using anti-polyubiquitin antibodies and immunofluorescence detection using an array scanner. Signal intensities were transformed and are displayed as heat-map. (B) Distribution plot of polyubiquitination changes of 3,873 in vitro polyubiquitinated proteins after Gsk3 inhibition by LiCl (Log2[Control/LiCl]). Arrays were treated with 30 mM LiCl or 30 mM NaCl (Control). LiCl reduced polyubiquitination of 864 proteins >1.8-fold. Some candidates, associated with cell cycle progression and selected for further validation are indicated in the plot. (C) Cluster of mitotic associated proteins that are significantly regulated by LiCl in B. The functional network was determined using String 9.1 and color-coded based on the fold change in the microarray (Dataset S1). (D) Western blots of indicated proteins from Xenopus eggs (naturally arrested in metaphase II) and released into interphase with the Ca2+ ionophore A23187 for 25 min, treated with LiCl, or mock treated (NaCl). β-cat, β-catenin; cycE, cyclin E; Interph., interphase; M. II, metaphase II; pH3, phosphorylated histone 3. (E) Western blots of stage VI oocytes in Prophase I and Progesterone-matured (Prog.) oocytes in metaphase II. Oocytes were injected (Inj.) with Morpholinos (MO) or mRNA as indicated. Co, control; dnWnt11, dominant negative Wnt11; pH3, phosphorylated histone 3; PPL, preprolactin (negative control).](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_6427/pmc04426427/pmc04426427__pnas.1423533112fig02.jpg)