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. Author manuscript; available in PMC: 2011 Jun 2.
Published in final edited form as: Nat Cell Biol. 2009 Dec 13;12(1):66–9. doi: 10.1038/ncb2006

Figure 2.

Figure 2

IpaH9.8 promotes NEMO ubiquitylation and degradation. (a) IpaH9.8–Flag bound to NEMO, as found in GST pulldown assays (left panel) and immunoprecipitation analysis of 293T cell lysates (center and right panel). (b) IpaH9.8 targets NEMO, and promotes its ubiquitylation. In vitro ubiquitylation assay with NEMO and a mixture of E1, UbcH5b, ATP and ubiquitin in the presence or absence of GST–IpaH9.8 or GST–IpaH9.8CA (left panel). Cells expressing Flag–NEMO, HA–Ub and Myc6–IpaH9.8 or Myc6–IpaH9.8CA were immunoprecipitated using an anti-Flag antibody and analysed for ubiquitylation by immunoblotting using anti-HA and anti-Flag antibodies. Whole cell lysates were immunoblotted with anti-Flag and anti-Myc antibodies (right panel). (c) Cells expressing Flag–NEMO, Myc6-IpaH9.8 and each of the HA-ubiquitin mutants as indicated were lysed and immunoprecipitated using an anti-Flag antibody. Ubiquitylated NEMO was analyzed by immunoblotting. (d) HeLa cells were transfected with Flag–NEMO and infected with Shigella strains. After infection, cell extracts were prepared at the indicated time points and subjected to immunoblotting. The remaining NEMO was quantified (graph). (e) HeLa cells were transfected with Flag–NEMO and infected with Shigella/pTBipaH9.8. After infection, cells were treated with DMSO, MG132 (40 μM) or E64D + pepstatin A, and cell extracts were prepared at the indicated time points. Samples were subjected to immunoblotting. The remaining NEMO was quantified (graph). WT, wild-type. IP, immunoprecipitate. IB, immunoblot. Ub, ubiquitin. For full scans of blots in a–d, see Supplementary Information, Fig. S7.