Gapdh

Cells were harvested and lysed with Cell Lysis Solution (Sigma-Aldrich). The supernatant was collected after centrifugation at 4°C (10,000 rpm) for 5 min. Total proteins were extracted and protein concentration was determined using the bicinchoninic acid assay (BCA) assay. Proteins (50 μg per lane) were separated using 12% SDS-PAGE. Proteins were then electrotransferred to a polyvinylidene fluoride (PVDF) membrane (Amersham Biosciences, Piscataway, USA). The PVDF membrane was rinsed with TBS for 10-15 min and placed in TBS/T blocking buffer containing 5% (w/v) skimmed milk powder. Then, it was incubated at 4°C overnight following the addition of an appropriate dilution of primary antibodies (1:2000 α-smooth muscle actin (α-SMA); 1:1000 collagen I; 1:2000 survivin; 1:2000 Bcl-2; 1:2000 Bax; 1:5000 GAPDH; all from Abcam, Cambridge, UK). The membrane was then rinsed with TBS-Tween 20 (TBST) 3 times and incubated with a horseradish peroxidase (HRP)-labeled goat anti-mouse IgG secondary antibody (1:50,000; Abcam) at room temperature for 1 h. After incubation, the membrane was rinsed 3 times with TBST. Protein bands were detected using an enhanced chemiluminescence kit (Perkin-Elmer, Waltham, USA) and quantified as a ratio to GAPDH. Quantification was performed using Imagequant LAS4000 (GE Healthcare, Tokyo, Japan).