Rabbit anti survivin

Podocytes were lysed with a RIPA buffer containing protein inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 μg/mL leupeptin and pepstatin). Thirty micrograms of the total protein were subjected to 8%–15% SDS-PAGE and transferred to nitrocellulose membranes (Amersham biosciences, Piscataway, NJ, USA). After blocking with PBS containing 5% nonfat dry milk for 1 h at room temperature, then incubated overnight at 4 °C with the following primary antibodies, rabbit anti-survivin (1:2000, Abcam, Cambridge, MA, USA), rabbit Anti-cleaved caspase 3 (1:750, Cell Signaling Technology, Beverly, MA, USA) and mouse Anti-GAPDH (1:5000, Chemicon, Temecula, CA, USA) antibody at 4 °C over night (14–16 h). Subsequently, the membranes were rinsed three times, each time for 10 min in PBS buffer with 0.05% Tween-20 and incubated with horseradish peroxidase-conjugated anti-rabbit or mouse IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA). After a final washing, the membranes were developed using an enhanced chemiluminescence reagent (Millipore, Bedford, MA, USA), and the specific protein bands were scanned and quantitated in relation to GAPDH. The densitometric analysis of images was performed using Image J software (National Institute of Mental Health, Bethesda, MD, USA).

Cells (HepG2 and SMMC-7721) treated with polydatin (1, 3, 10, 30, and 100 µM) for 48 h were lysed for 30 min in cold lysis buffer. After centrifugation at 12,000 g for 5 min at 4°C, the supernatant was collected as the total cellular protein extracts. Protein samples were separated on the 10% SDS-PAGE, and then transferred onto a polyvinylidene difluoride membrane. Membranes were incubated with 5% skimmed milk in TBST at room temperature for 1 h. Then, the membranes were incubated with rabbit anit-caspase-3 (1:1500), rabbit anti-caspase-9 (1:1500), rabbit anti-Bax (1:2000), rabbit anti-Bcl-2 (1:1000), rabbit anti-DKK-1 (1:1500), rabbit anti-beta-catenin (1:2000), rabbit anti-c-myc (1:1000), rabbit anti-cyclin D1 (1:2500), rabbit anti-survivin (1:1000), and rabbit anti-GAPDH antibodies (1:3000) (all from Abcam) overnight at 4°C, and washed three times with TBST. Then, the membranes were further incubated with appropriate HRP-linked secondary antibodies. The bands of specific proteins were visualized by western blotting Luminal Reagent (Thermo Fisher Scientific) according to manufacturer instructions.