Goat anti mouse or anti rabbit horseradish peroxidase conjugated secondary antibodies

The primary antibodies used were rabbit anti-MMP-2, -MMP-9 and -TIMP-2 (1:1,000). Horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit secondary antibodies (cat. no. 7076 and 7074; 1:10,000; Cell Signaling Technology, Inc.) were used to detect the primary antibodies on the western blots. Briefly, cells were incubated with PBS, BmK CT (5 µg/ml), Na¹³¹I or ¹³¹I-BmK CT (50 µCi/ml) for 72 h at 37°C. Total protein from the U87MG cells was isolated using radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology) and the protein concentration was measured using a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology). A total of 20 µg total protein was separated by 10% SDS-PAGE and transferred onto a polyvinylidene difluoride (PVDF) membrane. The PVDF membranes weres then blocked in PBS containing 5% skimmed milk and 0.1% Tween-20 for 2 h at room temperature. Subsequently, the membranes were incubated at 4°C overnight with the primary antibodies, followed by incubation with the corresponding secondary antibody for 1 h at room temperature. The bands were visualized with enhanced chemiluminescence reagents (Beyotime Institute of Biotechnology). The expression of the proteins was quantified using ImageJ software (version 1.52a; National Institutes of Health, Bethesda, MD, USA) and compared with that of the control (β-actin).