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. 2014 Jul 1;28(13):1429–1444. doi: 10.1101/gad.242131.114

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© 2014 Zheng et al.; Published by Cold Spring Harbor Laboratory Press

This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genesdev.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.

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Figure 6. — Differential binding of USP9x underlies control of FOXO3a stability by EglN2. (A) Immunoblot analysis of T47D cells that were infected with a lentivirus encoding the indicated USP9x shRNAs or Ctrl shRNA. (B) Immunoblot analysis of T47D cells that were infected with a lentivirus encoding HA-FOXO3a and then superinfected with a lentivirus encoding USP9x shRNA (364 or 064) or Ctrl shRNA. (C) Immunoblot analysis of 293T cells that were infected with a lentivirus encoding USP9x shRNA (364) or Ctrl shRNA and then, after drug selection, transfected with a plasmid encoding V5-tagged mouse full-length USP9x or empty vector (Ctrl). (D) Immunoblot (IB) assays of whole-cell extracts (WCE) and immunoprecipitates (IP) of T47D cells that were infected with a lentivirus encoding USP9x shRNA (sh128 or sh364) or Ctrl shRNA and then, after drug selection, transiently transfected with plasmids encoding Flag-ubiquitin and HA-FOXO3a followed by treatment with 10 μM MG132 for 16 h. (E) Immunoblot analysis of MCF-7 cells that were infected with a lentivirus encoding shRNA against either USP9x (364) or Ctrl shRNA followed by 100 μg/mL cycloheximide for the indicated duration. (F) Immunoblot analysis of 293T cells that were cotransfected with plasmids encoding V5-tagged USP9x (wild-type or C1556S) and HA-FOXO3a. (G) Immunoblot (IB) assays of whole-cell extracts (WCE) and immunoprecipitates (IP) of T47D cells that were transfected with a plasmid encoding HA-FOXO3a (wild-type or P426A;P437A) followed by treatment with 10 μM MG132 for 16 h. (H) Immunoblot analysis of bound USP9x recovered from lysed T47D cells that were incubated with 20 μL of Neutravidin-Sepharose preloaded with the indicated FOXO3a peptides. (I,J) Immunoblot analysis of T47D cells that were infected with a lentivirus encoding USP9x shRNA (364) or Ctrl shRNA and then, after drug selection, treated with the indicated drugs or siRNAs.