Using Western blot, CREB1 phosphorylation at Ser133 is detected in EGF-treated A431 cells (+), compared with untreated cells (-), whereas no change in total CREB1 levels was observed.
Using the CREB1 assay kits, CREB1 phosphorylation at Ser133 is detected in EGF-treated A431 cells (+), compared with untreated cells (-), whereas no change in total CREB1 levels was observed.
PhosphoTracer CREB1 (pS133) + total CREB1 ELISA Kit (ab119658) used in Sandwich ELISA. The results show that CREB is readily detectable in several cell lines.
A431 cells were seeded at 30K cells/well in medium containing 10% FBS in a 96 well microplate, and cultured overnight. The following day the culture medium was removed, and the cells were serum starved for 60 mins, before treating the cells with varying concentrations of forskolin for 30 min. The medium was removed from the wells, and the cells were lysed with 120 µL/well Lysis Mix, with shaking for 10 min. 50 µL of lysate was transferred to replicate wells of a PhosphoTracer assay plate, and Antibody Mix specific for either phospho CREB or total CREB (50 µL/well) was added to the lysates. The plates were incubated for 1 hr at room temp, with shaking. Plates were washed and Substrate Mix was added. The plates were covered in foil and incubated for 10 min with shaking. Signal in the wells was determined using a plate reader.