COP9 Integrity Is Essential for Its Function Figure 7. CAND1 is not required for regulation of the circadian rhythm. (A–C) Normal conidiation rhythms of cand1 mutants in dark–dark (A), light–dark (B), and temperature cycles (C) measured by race tube assays. At least four replicates were tested under each condition. Black lines indicate the growth fronts every 24 h. doi:10.1371/journal.pgen.1002712.g007 c-Jun in HeLa cells in which CSN-5 is downregulated is rescued equally by over-expression of the deneddylation mutant CSN- 5D151N or wild-type CSN-5 [20]. These data suggest that two activities of CSN may function parallel for regulating the activity of CRLs. Bennett et al. found that Cul1K720R (a constitutively unneddy- lated Cul1 mutant) assembles with CSN, SKP-1, and most F-box proteins to the same extent as wild-type Cul1 [51]. Our IP experiments also show that wild-type CSN interacts with both neddylated and unneddylated Cul4. These findings suggest that the CSN can interact with CRLs independent of the prior ned- dylation of cullins. In plants, genetic results also suggest that during early embryo development and germination, neddylation/ deneddylation cycling is not absolutely required, although it becomes more important during seedling establishment and later in development [21], suggesting that the CSN has distinct bio- chemical functions that orchestrate development in the appropri- ate spatial and temporal setting. Figure 8. CAND1 is not required for regulation of the SCFFWD-1 ubiquitin ligase. Western blot analyses showing degradation of FRQ (A) and FWD-1 (C) in wild-type and cand1KO strains after addition of cycloheximide (10 mg/mL). Densitometric analyses from four independent experiments showing the degradation of FRQ (B) and FWD-1 (D). Arrows point out FWD-1 protein bands. Asterisks indicate nonspecific bands detected by FRQ antibody (A) or FWD-1 antibody (C). doi:10.1371/journal.pgen.1002712.g008 PLoS Genetics | www.plosgenetics.org 11 May 2012 | Volume 8 | Issue 5 | e1002712