Western blot analysis of extracts from U937 cells treated with either LiCl or NaCl for the indicated times, using Phopho-C/EBPalpha (Thr222/226) Antibody (upper) and C/EBPalpha antibody (lower). C/EBPalpha phosphorylation at Thr222/226 is abolished by the specific GSK3 inhibitor LiCl, but not by NaCl, indicating that phosphorylation at these sites are depends on GSK3 kinase.
|MW (kDa)||30, 42, 45|
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-C/EBPα (Thr222/226) Antibody detects endogenous levels of C/EBPα only when phosphorylated at threonine 222 and 226. This antibody does not cross-react with other phosphorylated C/EBP isoforms.
Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Thr222/226 of mouse C/EBPα. Antibodies are purified by protein A and peptide affinity chromatography.
CCAAT/enhancer-binding proteins (C/EBPs) are a family of transcription factors that are critical for cellular differentiation, terminal function, and inflammatory response (1). Six members of the family have been characterized (C/EBPα, β, δ, γ, ε, and ζ) and are distributed in a variety of tissues (1). Translation from alternative start codons results in two isoforms of C/EBPα (p42 and p30), which are both strong transcriptional activators (2). It has been reported that insulin and insulin-like growth factor-I stimulate the dephosphorylation of C/EBPα, which may play a key role in insulin-induced repression of GLUT4 transcription (3). Phosphorylation of C/EBPα at Thr222, Thr226, and Ser230 by GSK-3 seems to be required for adipogenesis (4).
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