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Figure 7. ZC4H2 stabilizes Smad1/5 by attenuating their ubiquitination. (a,b) Smad1 and Smad5, but not Smad1-ΔLinker, were stabilized by ZC4H2 overexpression. FLAG-tagged Smad1, Smad5 or Smad1-ΔLinker and myc-tagged ZC4H2 plasmids were transfected into HEK293 cells as indicated. After 48 h, cell lysates were analysed by western blotting. (c–f) Effects of ZC4H2 overexpression on the stability of Smad1 (c,d) or Smad5 (e,f). HEK293 cells were transiently transfected with the indicated plasmids. At 36 h post-transfection, cycloheximide was added to all samples, and the cells were then harvested at the time points indicated. Levels of Smad1 or Smad5 were determined by western blotting with anti-FLAG antibody. In all cases, α-tubulin was used as a loading control. The relative levels of Smad1 or Smad5 were quantified densitometrically and normalized against α-tubulin (d,f). The data shown in panel (c,e) are the averages of three independent experiments. (g) Schematic diagram of the pGal4-TK-Luc reporter construct. Gal4 BE, Gal4 binding elements. (h) ZC4H2 enhanced the transcriptional activity of Smad1 and Smad5. Gal4-Luc reporter construct was transfected into HEK293 cells together with the combination of myc-tagged ZC4H2 and Gal4 fused Smad1/5 or ZC4H2, as indicated. The luciferase activity was measured at 48 h after transfection. n.s., not significant; **p < 0.01. (i,j) Effects of ZC4H2 overexpression on the polyubiquitination status of Smad1, Smad1-ΔLinker (i) or Smad5 (j) proteins. HEK293 cells were transiently transfected with the indicated plasmids and treated for 8 h with MG132 before harvesting. Smad proteins were coimmunoprecipitated with anti-FLAG antibody and then detected for polyubiquitin chains with the antibody against ubiquitin. WT, wild-type; WCL, whole-cell lysate; IB, immunoblot; IP, immunoprecipitation; CHX, cycloheximide. 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 Larger Image Printer Friendly View |