Xenbase Image ID: 158921

Figure 10. ZC4H2 mutants showed weaker Smad-stabilizing activity and might be involved in neural development in humans. (a,b) The effects of overexpression of various ZC4H2 mutants on the protein level of Smad1 (a) or Smad5 (b) in HEK293 cells. FLAG-tagged Smad1 or Smad5 and myc-tagged various ZC4H2 mutant plasmids were transfected into HEK293 cells as indicated. After 48 h, cell lysates were analysed by western blotting. (c,d) The effects of overexpression of various ZC4H2 mutants on the expression of Id1-Luc reporter induced by rhBMP2 treatment in Hep3B (c) and C2C12 (d) cells. Hep3B or C2C12 cells were transfected with the indicated plasmids together with Id1-Luc reporter plasmids and then treated with rhBMP2 for 24 h. Luciferase activity was measured at 48 h after transfection. RLU, relative light units; n.s., not significant; *p < 0.05 and **p < 0.01. (e,f) The association between Smad1 or Smad5 and various ZC4H2 mutants in co-IP assays in HEK293 cells. HEK293 cells were transiently transfected with different combinations of expression vectors of Smad1 or Smad5 and various ZC4H2 mutants as indicated, and treated for 8 h with MG132 before harvesting. Cell lysates were incubated with anti-FLAG beads, washed and subsequently analysed by western blotting. (g,h) The effects of overexpression of various ZC4H2 mutants on the polyubiquitination status of Smad1 or Smad5 in HEK293 cells. HEK293 cells were transiently transfected with the indicated plasmids and treated for 8 h with MG132 before harvest. Smad proteins were co-immunoprecipitated with anti-FLAG antibody and then detected for polyubiquitin chains with the antibody against ubiquitin. (i,j) In situ hybridization assays showed Sox2 expression in the embryos injected with control MO (25 ng), ZC4H2 MO (25 ng) or ZC4H2 MO (25 ng) together with various ZC4H2 mutant mRNAs (0.6 ng). LacZ mRNA was co-injected to trace the injected sides (stained red on the right sides). (j) Statistics of the data shown in panel (i). WT, wild-type; WCL, whole-cell lysate; IP, immunoprecipitation; IB, immunoblotting. Image published in: Ma P et al. (2017) © 2017 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 Gene Synonyms Species Stage(s) Tissue sox2.S anop3, LOC108718087, mcops3, Sox-2, XLSOX-2, Xsox-2, XSox2 X. laevis Throughout NF stage 17 neural plate Image source: Published Experiment + Assay Source Phenotypes and Disease Xla Wt + zc4h2 + zc4h2 MO + NF18 (in situ hybridization) Fig. 10 i Expression Phenotype increased amount sox2.S expression in neural plate mislocalised sox2.S expression in neural plate Larger Image Printer Friendly View

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