Anti pi3ca

After treatment with NP-Ø and NP-427, the FaDu seeded in 24-well plates were fixed with 4% paraformaldehyde (PFA) for 10 min. The cells were blocked for 1 h at 37 °C and incubated overnight at 4 °C with several antibodies: p-AKT-Ser473 (1/75), p-AKT-Thr308 (1/75), and Annexin-V (1/100) from Abcam (Abcam, Cambridge, UK) and p-PDK1-Ser241 and anti-PI3CA (1/100) from Invitrogen (Invitrogen Corporation y Applied Biosystems Inc., Carlsbad, United States). After, the cells were incubated with the secondary antibodies, i.e., Alexa Fluor 546-conjugated goat anti-rabbit antibody (1/250; Molecular Probes, Eugene, United States) or Alexa Fluor 488-conjugated goat anti-rabbit antibody (1/250; Molecular Probes, Eugene, United States). at 37 °C for 45 min. Then, the cells were stained with 300 nM of 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; Sigma-Aldrich, San Luis, CA, United States), reactive with fluorescent blue, which intercalates with DNA, for 5 min at 37 °C. Fluorescence was visualized using an Olympus BX51 microscope. The specificity was assessed by omitting the primary antibody. ImageJ, a free image-processing program, was used for quantitative analysis of the image.

At the end of the experiments, the FaDu cells were lysed in a buffer T-Per (Pierce, Appleton, WI, United States) with Complete™ Protease Inhibitor Cocktail (Roche, Basel, Switzerland). The cellular proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA, United States). The primary antibodies used in this study included EGFR (1/1000), p-AKT-T308 (1/5000), total AKT (1/5000), p-AKT-S473 (1/5000), and β-actin (1/2000, used as a loading control) from Abcam and phospho-PDK1-Ser241 (1/1000) and anti-PI3CA (1/1000) from Invitrogen. The immunoreactive bands were visualized using SuperSignal West Pico Chemiluminescent Substrate (ThermoFisher Scientific, Waltham, MA, United States). A densitometric analysis of the film was performed using AlphaFaseFC software (Alpha Innotech Corporation, San Leandro, CA, United States).