ab81283 staining AKT1 (phospho S473) in MCF7 cells treated with DAPT (ab120633), by ICC/IF. Decrease in expression of AKT1 (phospho S473) correlates with increased concentration of DAPT, as described in literature.The cells were incubated at 37°C for 24h in media containing different concentrations of ab120633 (DAPT) in DMSO, fixed with 4% formaldehyde for 10 minutes at room temperature and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab81283 (1/200 dilution) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 goat anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.
Kc167 cells were treated with varying concentrations of DAPT (ab120633) in DMSO for 16hr at 25°C; DMSO only was used as the negative control. The cells were further incubated for 30 minutes with 4mM EGTA in PBS (in the presence of DAPT), and were then lysed for analysis. To measure Notch activity, Notch targets E(spl)mß-HLH and E(spl)m3-HLH mRNA levels were assayed. Data shows the fold change of mRNA levels of E(spl)mß-HLH and E(spl)m3-HLH under different conditions, normalised to DMSO treatment (negative control). Notch activation by EGTA is abrogated by treatment with DAPT.