COP9 Integrity Is Essential for Its Function Figure 3. Point mutations do not disrupt integrity of the CSN or interactions of the CSN with Cul1 and Cul4. (A) Immunoprecipitation assays confirming interactions between different versions of Flag-CSN-5 and Myc-CSN-6. Wild-type strain and wild-type strain expressing Myc-CSN-6 were used as negative controls. (B) The Myc-CSN-5, Myc-CSN-5H127A, Myc-CSN-5H129A, or Myc-CSN-5D140N in csn-5KO properly incorporates into CSN complex. (C) Silver-stained SDS-PAGE showing the two-step purification of Myc-His-CSN-5, Myc-His-CSN-5H127A, Myc-His-CSN-5H129A, or Myc- His-CSN-5D140N in the csn-5KO strains. A wild-type strain was used as the negative control. CSN subunits identified by mass spectrometry analysis in products of Myc-His-CSN-5 or Myc- His-CSN-5H127A are indicated. Asterisks indicate the two IgG bands. (D) Immunoprecipitation assays confirming the interaction between different versions of Flag-CSN-5 and Myc-Cul1. (E) Immunoprecipitation assays showing the interaction between different versions of Myc-CSN-5 and endogenous Cul4. The asterisk indicates a nonspecific cross-reacted protein band recognized by our Cul4 antiserum. doi:10.1371/journal.pgen.1002712.g003 undetectable after 3 h of CHX treatment (Figure 6A and 6B). In the csn-5H127A strain, the detection of Myc-SCON-2 signal ex- tended to 6 h after CHX treatment (Figure 6A and 6B). FBP94 (NCU04785), another F-box–containing protein in N. crassa, can also interact with SKP-1 (data not shown). As shown in Figure 6C and 6D, FBP94 was quite stable in the wild-type strain and csn-5KO PLoS Genetics | www.plosgenetics.org 7 May 2012 | Volume 8 | Issue 5 | e1002712