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. 2015 Jun 30;10(6):e0131113. doi: 10.1371/journal.pone.0131113

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© 2015 Aerne et al

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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Fig 5 — (A) Sav binds to Herc4. Myc-tagged Sav was immunoprecipitated using a Myc antibody and bound Herc4-Flag protein detected using Flag antibody. Myc-tagged Drosophila Rep3 was used as control. (B) herc4 RNAi stabilises Sav protein levels. Left panel: endogenous Sav levels (arrow) in non-transfected S2 cells. Asterisk indicates a non-specific band detected by the anti-Sav antibody. Left bottom panel: quantification presented as ratio of Sav/Tub levels. Right panel: levels of transfected Myc-tagged Sav. Right bottom panel: quantification presented as ratio of Sav-Myc/Tub levels (C) herc4 RNAi reduces Sav ubiquitylation. Sav ubiquitylation assay of Myc-tagged Sav expressed in S2 cells, treated with dsRed RNAi, herc4 RNAi or sav RNAi. Ubiquitylation assay was performed in the presence of 5mM NEM to block deubiquitylating enzyme activity (D) Overexpression of Herc4 results in destabilisation and increased ubiquitylation of Sav, which is dependent on the active site cysteine. Sav ubiquitylation assay of Myc-tagged Sav, Sav∆N and Sav∆C co-expressing either Flag-Herc4 or Flag-Herc4C/A.