Figure 6. mFbxl21 is a clock-controlled gene. A. Schematics depicting the promoter region of the mouse Fbxl21 gene, E’ indicate non-canonical E-Box motifs, D-Box indicates a putative response element for the PAR-bZIP proteins. B. The mouse Fbxl21 gene is responsive to both CLK/BM1 and the PAR-bZIP transcription factors. Transfection conditions per well: 50 ng of Fbxl21 promoter reporter, 50 ng of b-Gal reporter and either 100 ng of mClock+100 ng of mBmal1 or 100 ng of each of the PAR-bZIP transcription factors and/or necessary amount of empty vector to make a final amount of 300 ng. Data in A/B are mean6SEM of one triplicate representative from 3 replicate experiments. Different letters above bars indicate significantly different groups (p,0.05). doi:10.1371/journal.pone.0003530.g006 Promoter reporter constructs and b-galactosidase reporter con- struct were used at 50 ng/well. Total transfected DNA amount was set to an equal amount between all conditions by addition of the corresponding empty vector. Luciferase assay was performed on the day following transfection. Briefly, cells were rinsed twice in cold PBS and lysed for 15 min in lysis buffer (25 mM Tris, 2 mM EDTA, 1 mM DTT (dithiothreitol), 10% glycerol and 1% Triton X-100). The luciferase assay was performed using a Luciferase assay system kit (Promega) and a Lumicount microplate luminometer (Packard). Results were normalised to b-galactosi- dase activity. Data (in relative luminescence units) represent fold induction once normalised to b-galactosidase. Experiments were repeated three to five times, each condition in triplicate. For WB experiments, variable amounts of DNA were transfected, never exceeding 4 mg/well. Experiments were done at least in triplicate. Western blot and immunoprecipitation Cells were plated in 6-well plates and transfected as described above. For protein synthesis inhibition experiments, 25 mg/ml cycloheximide (CHX; Sigma) was added 24 h post-transfection for 8, 2 or 0 h prior to harvesting. Cells were rinsed twice in cold PBS, resuspended in RIPA buffer (50 mMTris-HCl, pH 8, 150 mM NaCl, 1% Nonidet P40, 0.5% Na-deoxycholate, 0.1% SDS, 0.5 mM DTT, 1 mM PMSF and a cocktail of protease inhibitors (Complete Mini; Roche) and incubated for 30 min on ice. After vortexing, the extract was centrifuged for 1 min at 16000 g and supernatants used for Western blot (WB) or kept at 280uC. For Fbxl21 in the Circadian System Figure 7. A model for Fbxl21 function in the mammalian circadian clock. Transcriptional control (green dashed line) by CLK/ BM1 directs rhythmic expression of Cry1 and the PAR-bZIP and also has a modest effect upon Fbxl21. Transcription of Fbxl21 mostly depends on the PAR-bZIP proteins. The FBXL21 protein in turn controls degradation of CRY1 (red diamond-ended line) and therefore exerts a pivotal role in the control of CRY1-mediated inhibition of CLK/BM1 transcription (red diamond-ended line). doi:10.1371/journal.pone.0003530.g007 immunoprecipitation assay cells were harvested 48 h post- transfection in IP buffer (150 mM NaCl, 5 mM EDTA, 0.5% Nonidet P40, 50 mM Tris, pH 7.8, 1 mM PMSF and a cocktail of protease inhibitors (Complete Mini; Roche). An aliquot of supernatant was kept as input for western-blot analysis while the rest was incubated overnight at 4uC with anti-Myc affinity resin (Sigma) in IP buffer. The beads were then washed twice with IP buffer, resuspended in Laemmli buffer (50 mM Tris/HCl pH 6.8, 2% SDS, 0.1% Bromophenol Blue, 10% glycerol and 100 mM DTT), denatured at 95uC for 5 min and the supernatant was resolved on a 6% or 12% SDS polyacrylamide gel. Nitrocellulose membranes (Amersham) were then probed with antibodies directed against Myc (Roche) or Flag (Sigma). Anti-mouse horseradish peroxidase-conjugated antibody (Sigma) and the ECLH (enhanced chemiluminescence) detection reagent (Amer- sham) were used for revelation using Hyperfilm ECL (Amersham). RT-PCR on tissues RT-PCR was performed as described in the tissue collection and cloning section. Co-amplification of an 879 bp fragment of oCKId was used as a positive control. Omission of cDNA in the PCR mix was used as a negative control. Initial screening for Fbxl3 was performed using oligos O47C (GGGGTAATCTCCTTCAGG) and O48C (CTAGGACTTTGAGCGAGG) yielding 471 and 327 nt PCR fragments for Fbxl3fl and Fbxl3sv, respectively. Another PCR was performed using oligos O51C (GCAACTG- GAACCAGGTGG) and O52C (GGAAGTGAGTCTGTCCT- GG) yielding a 474 nt PCR product for Fbxl3sv only since O51C spans the splicing junction (the sequence corresponding to O51C in Fbxl3fl being GCAACTGGAACCAGGTAT prevents complete hybidization of the oligo hence polymerisation). Confirming specificity of the approach, no PCR product was obtained with O51C/O52C when the Fbxl3fl expression vector was used as a template while the expected 474 nt product was readily amplified from the Fbxl3sv expression construct. PCR screening for Fbxl21 was performed using oligos O49C (CCATCTCCAGTATGT- CAGC) and O50C (ATGCCAGTTCTCTCAGACC) yielding 386 and 298 nt PCR fragments for Fbxl21fl and Fbxl21sv, respectively. PLoS ONE | www.plosone.org 7 October 2008 | Volume 3 | Issue 10 | e3530