P53 9282

Cells were collected and lysed in lysis buffer [100 mM Tris-HCI (pH 6.8), 4% (m/v) sodium dodecylsulfonate (SDS), 20% (v/v) glycerol, 200 mM 2-mercaptoethanol, 1 mM phenylmethyl sulfonylfluoride and 1 g/ml aprotinin] for 30 min on ice. The lysates were separated using centrifugation at 4°C for 15 min at 3,913 × g. The total protein concentration in the supernatants was detected using the BCA Protein assay kit (Beyotime Institute of Biotechnology). Proteins (20 µg) were separated using 8–15% SDS-PAGE and subsequently transferred to nitrocellulose membranes. These were saturated with 5% milk in Tris-buffered saline and 1% Tween-20 (TBST) and incubated with the following primary antibodies in a diluent overnight at 4°C: p53 (9282), Bcl-2 (2876), Bax (2772), TGF-β (3711), TGF-βRII (11888), Smad3 (9513), p-Smad2/3 (8828) and GAPDH (2118) (all 1:1,000; Cell Signaling Technology). Membranes were washed three times with TBST and incubated with HRP-conjugated goat anti-rabbit IgG (E030120-01; EarthOx Life Sciences) for 1 h at room temperature, followed by washing four times with TBST. An enhanced chemiluminescence kit (GE Healthcare Life Sciences, Chalfont, UK) was added to the membrane and detection was performed using an Odyssey^® CLx Infrared Imaging system (LI-COR Biosciences, Lincoln, NE, USA).