Odyssey version 3

Protein extractions were performed as previously described^{44 (link),49} . Primary antibodies HAS2 (Santa Cruz Biotechnology, sc-34068) (1:200 dilution), HSL (Santa Cruz Biotechnology, sc-74489) (1:200 dilution), α Tubulin (Santa Cruz Biotechnology, sc-53030) (1:200 dilution), Actin (Sigma #A4700) (1:1000 dilution), Adiponectin (homemade) (1:1000 dilution) were used. Protein abundance was detected using the one of the following secondary antibodies: Goat anti-Mouse IRDye 680RD (Li-cor 926-68070), Goat anti-rabbit IRDye 800CW (Li-cor 925-32211) or Goat anti-Rat DyLight 800 (Thermo Fisher SA5-10024) at 1:10,000 dilutions. Antibody decorated membranes were then visualized on a Li-Cor Odyssey infrared scanner (Li-Cor Bioscience). The scanned data were analyzed using Odyssey Version 3.0 software (Li-Cor Bioscience).

After exposure to the test compounds (450 μg/mL GYII; 200 μg/mL DR2 and DR3; or 40 μg/mL fucoidan) for 36 h, the 4T1 cells were harvested and lysed with RIPA lysis buffer (Beyotime Biotechnology, Beijing, China) containing Protease Inhibitor Cocktail Set III (Calbiochem, San Diego, CA, USA). The cell lysates were centrifuged at 12,000 rpm for 15 min at 4 °C, and the supernatants were collected. The protein concentration was determined using the Pierce BCA Assay Kit (Thermo Scientific, Rockford, IL, USA). Equal amounts (45 μg/lane) of total protein were separated by 12% SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked with 5% BSA (Amresco, Solon, OH, USA) at room temperature for 1 h and incubated overnight at 4 °C with the following primary antibodies: anti-heparanase (1:500), anti-FGF-2 (1:500), anti-VEGF (1:1000), anti-ERK/p-ERK (1:1000), anti-PI3K/p-PI3K (1:1000), anti-AKT/p-AKT (1:1000), and β-actin (1:1000). After washing the membranes in Tris-buffered saline with 0.1% Tween-20 (TBST), the membranes were probed with secondary antibodies (1:10,000) for 1 h at room temperature. The signals were detected using an Odyssey Infrared Imaging System (Li-cor Biosciences, Lincoln, NE, USA). The relative density of the protein bands was measured by Odyssey version 3.0 software (LI-COR Biosciences). The experiments were repeated three times.