Sample experiment using ab128571 on MCF-7 cells treated with for 6 hours with Camptothecin or drug vehicle (DMSO). The data (presented as mean +/- SEM) were analyzed as described in Data Analysis section of the protocol.
Specificity of antibodies demonstrated by immuno-cytochemistry. Primary antibodies used in this assay kit were validated by staining MCF-7 cells treated with Camptothecin or vehicle and imaged by fluorescent microscopy. (1) Mostly nuclear localization of both p53 phospho S392 and total p53 protein signals, and (2) an increase in both p53 phospho S392 and total p53 protein signals upon Camptothecin treatment.
Antibody specificity demonstrated by Western Blot Analysis. Primary antibodies used in this assay kit were validated by Western Blotting using Hek293T induced with Etoposide (to increase the levels of p53 phosphorylation). Cell extract was treated with increasing concentrations of λ protein phosphatase (lane 2, 400x diluted; lane 3, 100x diluted; lane 4, 25x diluted) or left untreated (lane 1). Samples were analyzed by Western blotting using the rabbit p53 phospho S392 antibody (A). The membrane was re-probed with the mouse total p53 protein antibody (B).