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. 2007 Apr 9;177(1):51–61. doi: 10.1083/jcb.200611036

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Copyright © 2007, The Rockefeller University Press

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Figure 6. — Herp K63-linked polyubiquitination is POSH dependent and is enhanced by Tpg. (A) Analysis of POSH expression in H310 cells constitutively expressing a POSH shRNA and in H314 cells (control) expressing a scrambled shRNA. Detection of POSH was performed by Western blot analysis with anti-POSH (PT1). (B) Analysis of Herp ubiquitination in H310 cells. Cells were cotransfected with Herp-Flag and HA-Ub-K⁶³ either in the presence (+) or absence (−) of shRNA-resistant POSH. 24 h after the transfection, 2 μM Tpg was added to each culture, and the cells were further incubated for the indicated time periods, after which detergent lysates were prepared and Herp was isolated by immunoprecipitation with anti-Flag. For detection of Herp-Ub conjugates, the Herp immune complexes were resolved by SDS-PAGE (7.5% gels) and subsequently subjected to Western blot analysis with anti-HA (Ub; top) or with anti-Flag (Herp; bottom).