Horseradish peroxidase hrp labeled goat anti rabbit igg

Protein lysates were separated by 10% SDS-PAGE, and electrophoretically transferred to PVDF (polyvinylidene difluoride) membrane (Millipore). Then, the membrane was incubated with rabbit monoclonal antibody against human SNAI2, ZEB1, E-cadherin and vimentin (Cell Signaling Technology, USA) followed by HRP (horseradish peroxidase)-labeled goat-anti rabbit IgG (Santa Cruz Biotechnology, USA) and detected by chemiluminescence. GAPDH was used as a protein-loading control.

After cells were washed with ice-cold PBS, total proteins were extracted from cells using RIPA buffer supplemented with protease inhibitor cocktail on ice. Cell lysates were centrifuged at 14,000 rpm at 4 °C for 10 min. In total, 40 μg of total protein were separated by 10% SDS-PAGE and electrophoretically transferred to the PVDF membrane (Millipore Corporation, USA). The membranes were blocked with 5% fat-free milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1h at room temperature, followed by incubation with specific antibodies against TFPI-2 (Abcam, USA), caspase-3, cleaved caspase-3, caspase-8, cleaved caspase-8, caspase-9, cleaved caspase-9, and cleaved PARP (Cell Signaling Technology, USA) in 5% fat-free milk in TBST at 4 °C overnight. Membranes were washed and incubated for 2 h with HRP (horseradish peroxidase)-labeled goat-anti-rabbit IgG (Santa Cruz Biotechnology, USA), then detected, and visualized by chemiluminescence. Protein expression was analyzed by the ImageJ software and normalized to that of GAPDH (Cell Signaling Technology, USA).

Cells were homogenized with cold RIPA lysis buffer followed by 3 cycles of 20-s bursts of sonication at 4°C. The amount of protein per sample was determined using a dye-binding protein assay (Bio-Rad). Electrophoresis was performed on the samples using 4–12% or 10% acrylamide gels (Invitrogen) and transferred to polyvinylidene difluoride membranes (Millipore) by electroelution. Membranes were blocked in blocking solution [20 mM TBS Tween (1%) (TBST) containing 3% bovine serum albumin (BSA)] and then incubated with primary antibodies overnight at 4°C. After 3 washes with TBST, the membrane was incubated with secondary antibody for 1 h at room temperature (RT). Horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (Santa Cruz Biotech) was used as secondary antibody. After another 3 times wash with TBST to remove the rest secondary antibody, membrane was incubated with ECL reagent (Amersham Pharmacia Biotech, Piscataway, NJ, United States) and prepared for imaging. Bands were compared to molecular weight standards (Santa Cruz Biotech). GAPDH served as the internal reference by which to standardize the other protein bands. The calculated ratio of the control group was normalized to 100%, and the comparisons of other groups to the control group were represented as percentages.