Immunofluorescence analysis of CREB [pS129/pS133] was done on 70% confluent log phase HeLa cells. The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.25% Triton™ X-100 for 10 minutes, and blocked with 5% BSA for 1 hour at room temperature. The cells were labeled with CREB [pS129/pS133] Rabbit polyclonal Antibody (44297G) at 2 µg/mL in 1% BSA and incubated for 3 hours at room temperature and then labeled with Alexa Flour 488 Goat Anti-Rabbit IgG Secondary Antibody (A11008) at a dilution of 1:400 for 30 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (S36938). F-actin (Panel c: red) was stained with Alexa Fluor 594 Phalloidin (A12381). Panel d is a merged image showing nuclear localization. Panel e shows no primary antibody control. The images were captured at 20X magnification.
|Tested species reactivity||Human, Mouse|
|Host / Isotype||Rabbit / IgG|
|Immunogen||Phosphopeptide containing amino acid sequence RRP[pS]YRK, conjugated to KLH.|
|Purification||Antigen affinity chromatography|
|Storage buffer||Dulbecco's PBS, pH 7.3, with 1mg/ml BSA, 50% glycerol|
|Contains||0.05% sodium azide|
|Tested Applications||Dilution *|
|ChIP assay (ChIP)||10µl|
|Western Blot (WB)||1:200-1:2000|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
Cyclic AMP response element binding protein (CREB) is a basic/leucine zipper transcription factor that binds the cyclic AMP response element (CRE) and activates transcription in response to a variety of extracellular signals including neurotransmitters, hormones, membrane depolarization, and growth and neurotrophic factors. Activation of CREB is dependent upon the phosphorylation of serine 133. Phosphorylation occurs via p44/42 MAP kinase and p90RSK and also via p38 MAP kinase and MSK1. Although CREB will bind DNA independent of its phosphorylation state, only the phosphorylated form is competent as a transcription factor. CREB binding protein (CBP), a transcriptional coactivator that directly interacts with CREB, binds to CREB in the region of serine 133. CREB appears to play an important role in learning and memory.
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