Western blot analysis of extracts of COS cells untransfected (lane 1), or transfected with wild-type mouse C/EBPalpha (lane 2), S21A (lane 3), and S21D (lane 4) mutants, using Phospho-C/EBPalpha (Ser21) Antibody (upper) or C/EBPalpha Antibody (lower). (Provided by Dr. Hanna Radomska, Beth Israel Deaconess Medical Center, Boston, MA).
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.
Confocal immunofluorescent analysis of THP-1 (left) and Jurkat (right) cells using C/EBPα (D56F10) XP® Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
Western blot analysis of extracts from adipocytes (differentiated 3T3-L1) treated with insulin for the indicated times, using Phospho-C/EBPbeta (Thr235) Antibody.
Western blot analysis of extracts from COS cells, untransfected or transfected with human or mouse C/EBPbeta (LAP), using C/EBPbeta (LAP) Antibody.
Western blot analysis of extract from differentiated 3T3-L1 cells, using C/EBPδ Antibody.
Western blot analysis of extracts from C2C12 cells, untreated or tunicamycin-treated (2 μg/ml, 8 hr), using CHOP (D46F1) Rabbit mAb (upper) or β-Actin Antibody #4967 (lower).
Western blot analysis of extracts from mouse adipocytes treated with insulin for the indicated times, using Phospho-C/EBPalpha (Ser21) Antibody.
Confocal immunofluorescent analysis of differentiated 3T3-L1 cells using C/EBPα (D56F10) XP® Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
Western blot analysis of extracts from NIH/3T3-L1, differentiated for the indicated times, using C/EBPbeta (LAP) Antibody.
Immunohistochemical analysis of paraffin-embedded mouse lung using C/EBPα (D56F10) XP® Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded cell pellets, THP-1 (left) or Jurkat (right), using C/EBPα (D56F10) XP® Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded human hepatocellular carcinoma using C/EBPα (D56F10) XP® Rabbit mAb in the presence of control peptide (left) or antigen-specific peptide (right).
Immunohistochemical analysis of paraffin-embedded human tonsil using C/EBPα (D56F10) XP® Rabbit mAb.
Western blot analysis of extracts from Hep G2 and LNCaP cells using C/EBPα (D56F10) XP® Rabbit mAb.
|Phospho-C/EBPα (Ser21) Antibody 2841||20 µl||
|Phospho-C/EBPα (Thr222/226) Antibody 2844||20 µl||
||H M||30, 42, 45||Rabbit|
|C/EBPα (D56F10) XP® Rabbit mAb 8178||20 µl||
||H M||42, 28||Rabbit IgG|
|Phospho-C/EBPβ (Thr235) Antibody 3084||20 µl||
||H M||19 LIP. 36 LAP. 38 LAP.||Rabbit|
|C/EBPβ (LAP) Antibody 3087||20 µl||
||H M||35 to 38 mouse LAP. 45 to 49 human LAP.||Rabbit|
|C/EBPδ Antibody 2318||20 µl||
|CHOP (D46F1) Rabbit mAb 5554||20 µl||
|Anti-rabbit IgG, HRP-linked Antibody 7074||100 µl||
The C/EBP Antibody Sampler Kit provides an economical means of evaluating the C/EBP family of transcription factors and several phosphorylation sites that are involved in its activation. The kit includes enough antibody to perform two western blot experiments with each primary antibody.
Unless otherwise indicated, each antibody will recognize endogenous total levels of their target protein. Each activation state antibody recognizes the phosphorylated form of its target. Phospho-C/EBPβ (Thr235) Antibody recognizes endogenous levels of human liver activating protein (LAP) only when phosphorylated at Thr235, mouse and rat LAP only when phosphorylated at Thr188, and liver inhibitory protein (LIP) only when phosphorylated at Thr37. The C/EBPβ (LAP) Antibody detects endogenous levels of total C/EBPβ, the p38 and p36 LAPs, but not the p20 LIP.
Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser21 of human C/EBPα, Thr222/226 of mouse C/EBPα, or Thr235 of human C/EBPβ. Polyclonal antibodies are also produced by immunizing animals with a synthetic peptide corresponding to the amino-terminal sequence of human C/EBPβ or the sequence of mouse C/EBPδ. Polyclonal antibodies are purified by protein A and peptide affinity chromatography. Monoclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ala176 of human C/EBPα protein or to residues surrounding Leu159 of human CHOP protein.
CCAAT/enhancer-binding proteins (C/EBPs) are transcription factors critical for cellular differentiation, terminal function, and inflammatory response. Six characterized family members (C/EBPα, β, δ, γ, ε, and ζ) are distributed in a variety of tissues (1). Translation from alternative start codons results in two C/EBPα isoforms (p42 and p30) that are strong transcriptional activators (2). Research studies indicate that insulin and insulin-like growth factor-I stimulate C/EBPα dephosphorylation, 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 may be required for adipogenesis (4). The two forms of C/EBPβ, 38 kDa liver activating protein (LAP) and the 20 kDa liver inhibitory protein (LIP), may result from alternative translation. The 38 kDa LAP protein is a transcriptional activator while LIP may inhibit C/EBPβ transcriptional activity (5). Phosphorylation of C/EBPβ at distinct sites stimulates its transcriptional activity (6-8). Phosphorylation at the rat-specific site Ser105 in C/EBPβ appears essential for C/EBPβ activation in rat (9). C/EBPδ protein is highly expressed in adipose tissue, lung, and intestine (10). Increased expression of C/EBPδ mRNA levels during adipogenesis suggests that C/EBPδ plays an important role in positively regulating adipogenesis (10,11). C/EBPδ is expressed in the mammalian nervous system and plays an important role in long-term memory (10,12). CHOP is a C/EBP-homologous protein that inhibits C/EBP and LAP in a dominant-negative manner (13). CHOP expression is induced by cellular stresses, including starvation; induced CHOP suppresses cell cycle progression from G1 to S phase (14). During ER stress, the level of CHOP expression is elevated and CHOP functions to mediate programmed cell death (15).
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