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. Author manuscript; available in PMC: 2012 Nov 1.
Published in final edited form as: Nat Med. 2012 Apr 15;18(5):774–782. doi: 10.1038/nm.2728

Figure 2. Influence of transcriptional, translational or post-translational mechanisms on Period 2 protein levels.

Figure 2

(a) Synchronized HMEC-1 were treated with vehicle, or ADORA2B agonist BAY 60-6583 and blotted after indicated time periods; one of three representative experiments is displayed, and quantified below (*p<0.05, n=3). (b,c) Synchronized HMEC-1 treated with ADORA2B agonist (10mM) or forskolin (30mM) with and without actinomycin (ACT, 2µM, b) or cycloheximide (CXM, 40 µg/ml, c). Effectiveness of ACT or CXM are shown in Supplementary Fig S12 a,b. (d, left) Proposed model of adenosine-dependent alteration in post-translational PER2 protein stability; ADORA2B: A2B adenosine receptor. (d, right) PER2 protein levels following inhibition of proteasomal degradation (AM114 (10 µM; one of three representative blots is displayed). (e) Synchronized HMEC-1 were treated with vehicle, or ADORA2B agonist BAY 60-6583 and protein lysates were isolated for native protein complexes using a PER2 antibody covalently coupled (immobilized) onto an amine-reactive resin. Immunoprecipitated protein was analyzed using immunoblot against ubiquitin. One representative blot of three is displayed. (e, middle, right) Changes in protein shown by densitometry (n=3). (f) Synchronized HMEC-1 treated with vehicle, or ADORA2B agonist BAY 60-6583 and blotted for total CUL1 or neddylated CUL1 using a specific CUL1 antibody on a gradient gel. (g) HMEC-1 at 6h following synchronization treated with ADORA2B agonist BAY 60-6583 alone (10 µM), or following additional pre-treatment with ADORA2B antagonist PSB 1115 (1 µM) and blotted for neddylated CULLIN using a NEDD8 antibody (one of three representative experiments is displayed). (h, i) HMEC-1 following siRNA repression of CSN5 (siCSN5) or treatment with non-specific control siRNA (csiR) were synchronized by serum starvation, lysed and blotted for neddylated CULLIN (h) or PER2 (i) at indicated time points (one of three representative experiments is displayed). (j) Cardiac myocytes were isolated from wild-type (WT) or Adora2b−/− mice, exposed to hypoxic preconditioning (HPC) or control conditions and blotted for Per2 or neddylated Cullin (one representative blot of three independent experiments is displayed, one animal per experiment).