Complete protease inhibitor mixture

The cells in the two groups were collected on third and fifth days of co-culture, homogenized in 1X cell lysis buffer (Solarbio) supplemented with 1 mM PMSF and complete protease inhibitor mixture (Solarbio) for 30 min on ice, and then centrifuged at 11,000 rpm for 10 min at 4°C. The supernatant was measured using a Bicinchoninic Acid Protein Assay kit (Solarbio). The measurement samples were combined with 5X SDS loading buffer and boiled at 100°C for 10 min. For each protein, equal amounts of samples (20–100 µg) from each group were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis as previously described (26 (link)). After proteins were transferred onto a polyvinylidene difluoride membrane, the membrane was incubated with 5% BSA at room temperature for 2 h to block the nonspecific protein site and then corresponded with primary antibodies [TRAIL from Abcam; DR4 and DR5 from Abcam; vascular endothelial growth factor receptor 2 (VEGF-R2), caspase-8 and −3, and PARP from Cell Signaling Technology, Inc. (Danvers, MA, USA); β-actin from ZSGF-BIO] at 4°C overnight. This step was followed by incubation with HRP-conjugated secondary antibodies (ZSGF-BIO). Visualization was achieved using a SuperSignal West Pico Trial kit (Thermo Fisher Scientific, Inc.).

The cells from co-culture were homogenized in cell lysis buffer (Solarbio), supplemented with 1 mM phenylmethanesulfonyl fluoride (PMSF) and complete protease inhibitor mixture (Solarbio). The homogenate was centrifuged at 11,000 rpm for 15 min at 4°C. The supernatant was incubated with anti-TRAIL (Abcam) crosslinked beads at 4°C overnight with rotation. The Pierce Crosslink Magnetic IP kit instruction was used for the pretreatment of beads and immunoprecipitation. The associated proteins were detected using western blot analysis. Homogenates from co-culture cells were used as positive controls.