Enhanced chemiluminescence detection reagent kit

Total cell extracts were prepared from sub-confluent cells using Nonidet P-40 lysis buffer (20 mM HEPES-KOH [pH 7.6], 100 mM KCl, 5 mM MgCl₂, 2 mM dithiothreitol, 0.25% Nonidet P-40, 2 μg/ml leupeptin, 2 μg/ml pepstatin, and 102 μg/ml cycloheximide). Lysates were mixed with 2X SDS-loading buffer containing 10% β-mercaptoethanol, boiled for 5 min, and loaded onto 12% SDS-polyacrylamide gel for electrophoretic separation. The proteins were transferred onto a nitrocellulose membrane (Bio-Rad Laboratories, CA) at 300 milliamps for 40 minutes at room temperature (RT). Membranes were then blocked for nonspecific binding with 5% non-fat dry milk in TBST (TBS with 0.1% Tween 20; pH 7.4) for 1 hour, and incubated with the primary antibodies overnight at 4ºC. The following monoclonal and polyclonal antibodies were used: anti-FOXO1 and anti-FOXO3a (Cell Signalling, USA); anti-MiTF and anti-Smad1 (abcam, USA): anti-β-Tubulin (Sigma). After washing with TBST, membranes were incubated with species-specific secondary antibody conjugated to horseradish peroxidase enzyme for 1 hour at RT, and washed three times with TBST. Proteins were detected using an enhanced chemiluminescence detection reagent kit (Amersham Biosciences Inc.) and Kodak X-OMAT-AR film for autoradiography.