A dilution series of extract in Incubation Buffer in the working range of the assay. The extract was prepared from pellets of Hek293T cells treated with etoposide (ab120227).
Cells were treated with calyculin A + okadaic acid (Cal A + OA), etoposide (ab120227), camptothecin or drugs’ vehicles, as indicated. Diluted cell extracts were analyzed ab156027 (A, C) and p53 protein ELISA (B, D), using ab117995. Extract of Hek 293T treated with etoposide was used for positive control sample standard curves. Relative levels interpolated from standard curves and expressed in percent of etoposide- (A, B) and camptothecin- (C, D) treated samples are shown.
Extracts of Hek 293T cells (induced with etoposide) were treated with increasing concentrations of λ protein phosphatase (400x= 400-times diluted, 100x=100-times diluted, 25x=25-times diluted), or left untreated (Contr), and relative phospho Ser15 levels were determined using this kit. Dilutions of extracts of Hek 293T cells treated with etoposide were used to construct the standard curve.
Hek293T cells were treated with vehicle (lane 1) or etoposide (lanes 2-6). MCF7 cells were treated with vehicle (lane 7) or camptothecin for 6 (lane 8), 16 (lane 9) and 24 (lane 10) hours. Extracts of Hek293T cells (induced with etoposide) were treated with increasing concentrations of λ protein phosphatase (lane 4, 400x diluted; lane 5, 100x diluted; lane 6, 25x diluted) or left untreated (lane 3). Samples (lanes 1-2 and 7-10, 20 µg/lane; lanes 3-6, 8 µg/lane) were analyzed by Western blotting using the p53 pSer15 Detector Antibody of ab156027 kit (A), or a p53 antibody (ab1101) was used to detect total p53 protein (B).