Xenbase Image ID: 83166

Figure 6. Dzip1 prevents ubiquitin/proteasome-dependent degradation of Spop/HIB. (A) Spop is ubiquitinated in vivo. MT-Spop was injected into Xenopus embryos. Spop protein was purified by immunoprecipitation using an anti-myc antibody. Lysates and immunoprecipitates from injected and uninjected embryos were subjected to Western blot with the anti-myc antibody (left panel) and anti-ubiquitin antibody (right panel). The unmodified MT-Spop band migrated to 68 kDa. A smear of high molecular weight proteins (*), migrating slower than unmodified MT- Spop, could be detected in both myc and ubiquitin Westerns. (B) MT-Spop was injected into control or embryos that had received a prior injection of DMO. Animal caps were dissected at stage 9. Subsequently, caps were cultured in media with or without 2μg of lactacystin. Animal caps were harvested at stage 15 and subjected to Western blot to detect the levels of Spop. (C) Western blot results showing severe reduction of Flag-dDzip1 by dDzip1 dsRNA in S2 cells. �- tubulin serves as a loading control. (D) Dzip1 knockdown promoted proteasome-dependent degradation of HIB in S2 cells. S2 cells were cotransfected with Flag-HIB and GFP constructs and treated with dDzip1 dsRNA and/or MG132. Cell extracts were subjected to Western blot with the anti-Flag antibody to detect the levels of HIB. GFP serves as transfection and loading control. (E) Dzip1 knockdown promotes Ci stability in S2 cells. S2 cells were transfected with Flag-Ci and treated with dDzip1 dsRNA. Western blots were carried out with the cell lysates to determine the levels of Ci. Tubulin serves as a loading control. Image published in: Schwend T et al. (2013) Copyright © 2013. Image reproduced with permission of the publisher 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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