Hrp ecl system

Hep‐2 and TU212 cells in logarithmic phase were inoculated at 6 × 10⁵ cells/dish in 100 mm × 20 mm dishes and allowed to grow for 48 hours. After incubation with 0, 10, 30 and 50 μg/mL SM‐BFRE for 24 hours, the cells were collected by centrifugation and lysed in radio immunoprecipitation assay (RIPA) lysis buffer (Beyotime) to extract the proteins. The supernatant containing the protein was collected, and the concentration of the protein was measured by the bicinchoninic acid (BCA) kit (Beyotime). After denaturation, the protein was stored in a −80°C freezer until required. Protein extraction from the tumour tissues was performed following the same protocol. Equivalent amounts of the protein (40 µg) were separated by SDS‐PAGE gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes (Bio‐Rad). The membranes were blocked by 5% skim milk formulated with tris‐buffered saline with 0.5% Tween 20 (TBST) for 2 hours at room temperature. The membranes were then incubated with the primary antibody at 4°C overnight and incubated with the secondary antibody at room temperature for 1‐2 hours. The HRP ECL system (Bio‐Rad) was used to detect the protein band, and the grey value was calculated by Image Lab software.

Throat cancer cells were plated in the medium and allowed to cultivate until about 70−80% fusion. After being treated with four concentration gradients (0, 5, 10 and 20 μg/mL) of SD-BFRE for 24 h, the cells were separated and extracted in a RIPA lysis buffer (Beyotime, Shanghai, China). The supernatant was collected and its concentration was measured by the BCA kit (Beyotime, Shanghai, China). Next, the protein was put in slightly boiling water for 10 minutes, then stored in an −80 °C freezer for further analysis. The collection and extraction of tumor tissue protein was performed as mentioned previously. Equivalent amounts of the protein (40 µg) were parted by SDS-PAGE (10% or 12.5%) and then transferred to PVDF membranes (Bio-Rad, CA, USA) by electrophoresis. The membranes containing bands of protein were blocked with 5% skim milk powder dissolved in TBST for 2 h. After processing, the membranes were incubated with the primary antibody at 4 °C overnight. After being washed three times the next day, the membranes were incubated with the corresponding secondary antibody for 1 h 40 min. The HRP ECL system (Bio-Rad, CA, USA) was applicable to show the protein band. Meanwhile, the variation tendency of protein was analyzed by Image Lab software.