Western blot analysis of extracts from 293 cells, either untransfected or transfected with DYKDDDDK-tagged Rev-erbα expression constructs, using Phospho-Rev-erbα (Ser55/Ser59) Antibody (upper) or DYKDDDDK Tag Antibody (Binds to same epitope as Sigma's Anti-FLAG® M2 Antibody) #2368 (lower). The phospho-specificity of the antibody was verified by transfecting cells with a mutant DYKDDDDK-Rev-erbα (Ser55/59Ala) expression construct and by incubating 293/Rev-erbα (WT) extracts with (+) or without (-) λ phosphatase.
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. Store at –20°C. Do not aliquot the antibody.
For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody product webpage for recommended antibody dilution.
From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised June 2020
Protocol Id: 10
Phospho-Rev-erbα (Ser55/59) Antibody detects transfected levels of Rev-erbα protein when phosphorylated at Ser55/59. The antibody does not cross-react with other nuclear receptor proteins, including Rev-erbβ.
Mouse, Rat, Bovine
Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser55 and Ser59 of human Rev-erbα. Antibodies are purified by protein A and peptide affinity chromatography.
Reverse orientation c-erbA gene α (Rev-erbα, EAR-1, or NR1D1) is a widely expressed member of the orphan nuclear receptor family of proteins (1). Rev-erbα is highly expressed in adipose tissue, skeletal muscle, brain and liver, and regulates cellular proliferation and differentiation. Expression increases during differentiation in adipocytes and ectopic expression of Rev-erbα potentiates the adipocyte differentiation of 3T3-L1 cells (2). In addition, expression oscillates with circadian rhythm in liver cells and Rev-erbα regulates expression of BMAL1, ApoA-I and ApoC-III, all key regulators of circadian rhythm (3-7). Phosphorylation of Rev-erbα Ser55 and Ser59 by GSK-3β appears to stabilize Rev-erbα protein levels and is important for synchronizing and maintaining the circadian clock (8). Rev-erbα also regulates inflammation by targeting the NF-κB responsive genes IL-6 and COX-2 (9). Rev-erbα lacks the activation function 2 domain required for ligand-dependent activation of transcription by other members of the nuclear receptor family; thus it behaves as a constitutive repressor protein, recruiting the nuclear receptor co-repressor (N-CoR)/HDAC3 complex to target genes to repress transcription (10).
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