Human/Mouse/Rat Phospho-CREB (S133) DuoSet IC ELISA

Standard Curve

Specificity of Human/Mouse/Rat Phospho-CREB (S133) DuoSet IC ELISA Shown by Peptide Competition.

A lysate prepared from HeLa human cervical epithelial carcinoma cells treated with 200 nM PMA for 20 minutes was analyzed with this DuoSet IC ELISA. The Human/Mouse/Rat Phospho-CREB (S133) DuoSet IC Detection Antibody was either untreated (no peptide) or pre-incubated with a phosphopeptide containing the CREB S133 phosphorylation site, a phosphopeptide containing the GSK-3 beta S9 phosphorylation site, a phosphopeptide containing the c-Jun S63 phosphorylation site, or a phosphopeptide containing the Raf1 S338 phosphorylation site. Peptides were used at 40 ng/mL. Only the phosphopeptide containing the CREB S133 phosphorylation site blocks the signal, indicating that the DuoSet IC ELISA is specific for CREB phosphorylation at S133.

Quantification of Phosphorylated CREB in PMA-treated Human HeLa Cells.

Lysates prepared from HeLa human cervical epithelial carcinoma cells either uninduced or induced with 200 nM PMA for the indicated times were quantified with this DuoSet IC ELISA. The same lysates were also immunoblotted (inset) with either Anti-Human Phospho-CREB (S133) (Catalog # AF2510) or Anti-Human CREB (Catalog # AF2989) Polyclonal Antibodies. The DuoSet IC ELISA results correlate well with the amounts of phosphorylated CREB detected by Western blot. The immunoblot with anti-CREB antibody indicates that total levels of human CREB remained constant during incubation with PMA.The quantification of phosphorylated CREB with this DuoSet IC ELISA was also determined using cells pretreated with two concentrations of the MEK inhibitor U0126, which indirectly inhibits phosphorylation of CREB at S133.