ZNF395 Is an Activator of a Subset of IFN-Stimulated Genes

Figure 6

ZNF395 contains at least two independent IKK-dependent degradation motifs. (a) WB with extracts from RTS3b cells which have been transfected with vectors expressing two central fragments of ZNF395, Δ3, encoding amino acids 189–378 and Δ4, encoding amino acids 223–378, or Δ3 with mutations within the putative phosphodegron (lanes 9–12) as indicated in the figure. The cells were treated with poly I:C (lanes 2, 4), TNFα (lanes 6, 10), BMS-345541 (lanes 7, 11), or BMS-345541 and TNFα (lanes 8 and 12) as indicated. In lanes 15–17 the extracts have been incubated with λ-phosphatase (λ-ph) prior to analysis. The amino acid sequence from pos. 189–223 is given and the putative phosphodegron motif is indicated as well as mutations present in ZNF395Δ3 4xST/A or ZNF395 4xST/A. (b) RTS3b cells have been transfected with an expression vector for wt ZNF395 or ZNF395 4xST/A containing the mutations shown in (a) and treated with TNFα and BMS-345541 as indicated. (c) Expression vectors for ZNF395 and deletion mutants, which are depicted in the figure, were transfected into RTS3b cells which have been incubated with pI:C 24 h prior to harvesting. The WB was developed with the FLAG antibody since the anti-ZNF395 antibody recognizes only an internal fragment of ZNF395 (data not shown). The lower part shows a coimmunoprecipitation with extracts from cells transfected with expression vectors for FLAG-ZNF395, HA-IKKβ, and HA-IKKα. (EV = empty vector). indicates the position of the full length ZNF395 version.

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