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. 2014 Aug 8;33(20):2332–2348. doi: 10.15252/embj.201488447

Figure 6. Akirin2 interacts with IκB-ζ via the C-terminal conserved region.

Figure 6

A Schematic representation of Akirin2 conserved regions and mutant proteins used.

B Flag-IκB-ζ and/or Myc-Akirin2 were transfected in HeLa cells, and cell lysates were immunoprecipitated with anti-Myc Ab followed by immunoblot with anti-Flag and anti-Myc Ab.

C HeLa cells were transfected with Flag-Akirin2 plasmid. The cells were then stimulated with IL-1β for 12 h followed by the preparation of the cell lysates, and immunoprecipitation with anti-Flag Ab followed by immunoblot with anti-NF-κB p50, anti-NF-κB p65, anti-IκB-ζ, and anti-Flag Ab.

D PECs from LysM-Cre+; Akirin2fl/+ and LysM-Cre+; Akirin2fl/fl mice were treated with LPS (1 μg/ml) for 4 h. Cell lysates were collected and subjected to immunoprecipitation using anti-IκB-ζ Ab followed by immunoblot with anti-Akirin2 and anti-IκB-ζ Ab. PECs from IκBζ+/− and IκBζ−/− mice were harvested, and cell lysates were subjected to immunoprecipitation using anti-IκB-ζ Ab followed by immunoblot with anti-Akirin2 and anti-IκB-ζ Ab.

E Immunofluorescent staining of HeLa cells expressing Flag-Akirin2 mutants (green) together with DAPI (blue).

F HEK293 cells were transfected with indicated Myc-tagged Akirin2 mutant plasmids and Flag-tagged IκB-ζ. Cell lysates were collected and subjected to immunoprecipitation with anti-Flag Ab followed by immunoblot with anti-Myc and anti-Flag Ab.

G HeLa cells transfected with indicated Akirin2 mutant plasmids were stimulated with IL-1β for 12 h followed by the preparation of the cell lysates. Then, immunoprecipitation with anti-IκB-ζ Ab was performed followed by immunoblot with anti-Flag Ab.

H HEK293 cells were transfected with indicated Flag-tagged IκB-ζ mutant plasmids and Myc-tagged Akirin2. Cell lysates were collected and subjected to immunoprecipitation with anti-Flag Ab followed by immunoblot with anti-Myc and anti-Flag Ab.

I HEK293 cells were transfected with indicated Flag-tagged Akirin2 mutant plasmids and Myc-tagged BAF60a. Cell lysates were collected and subjected to immunoprecipitation with anti-IκB-ζ Ab followed by immunoblot with anti-Myc, anti-Flag, and anti-IκB-ζ Ab.

Data information: The results are representative of at least three independent experiments.

Source data are available online for this figure.