Anti mmp9

BK and anti-MMP-2 antibodies were obtained from Abcam (Cambridge, UK). The BK B2 receptor-specific antagonist, HOE-140, was purchased from Shanghai Top-Peptide Biotechnology Company (Shanghai, China). Recombinant human TGF-β1 was obtained from PeproTech Inc. (New Jersey, USA). Antibodies to fibronectin, Akt, and phosphorylated Akt (p-Akt) were purchased from Bioworld (Minnesota, USA), and antibodies to p-Erk1/2 and anti-MMP-9 were obtained from Cell Signaling Technology (Boston, USA).

B16-F10 cells (2 × 10⁶ cells per well) were seeded into a 10 cm dish for 24 h, and then cells were treated with different concentrations of PSS (12.5, 25, 50, 100 μg/mL) for 24 h. The medium was removed, and the cells were washed with PBS three times. Cells were then lysed in 200 μL of lysis buffer on ice. The total protein was determined using the Bicinchoninic Acid (BCA) Kit (Solarbio, Beijing, China). Equal amounts of protein in the cell extracts were fractionated by 10% SDS-PAGE, and then electrotransferred onto polyvinylidene fluoride (PVDF) membranes. After blocking with TBST (20 mM Tris-buffered saline and 0.1% Tween) containing 5% nonfat dry milk for 1 h at room temperature, the membranes were incubated for 2 h with monoclonal antibodies, such as anti-MMP-9, anti-MMP-2, anti-E-cadherin, anti-Vimentin, anti-ERK 1/2, anti-p-ERK 1/2, anti-AKT, anti-p-AKT (Ser473), anti-p38, anti-p-p38, anti-p-p38, anti-NF-κB, anti-p-NF-κB, and anti-β-actin, which were purchased from Cell Signaling Technology. The membranes were then washed three times and incubated with HRP-conjugated secondary antibodies (Abcam, Cambridge, Massachusetts, USA). The proteins were then detected using chemiluminescence agents (Amersham ECL, GE Healthcare, Buckinghamshire, UK).