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Recombinant TSP1 proteins

Protein production was carried out by Y-Biologics (Daejeon, South Korea). Briefly, sequences encoding mouse N-TSP1 (aa 19–259), middle fragment of TSP1 (M-TSP1; aa 260–690) and C-terminal fragment of TSP1 (C-TSP1; aa 691–1170) were ordered as a synthesized product (GeneArt, Thermo Fisher Scientific) and cloned into a Y-Biologics expression vector containing an N-terminal IgG Fc tag, such that each fragment was expressed as an Fc fusion recombinant protein. Each recombinant protein was targeted for secretion by the addition of the signal peptide sequence MGWSYIILFLVA TAADVHS at its N-terminus. FreeStyle 293-F Cells (R79007, Thermo) were used to produce recombinant proteins. Each DNA construct was transfected into HEK293F cells and cultured in a shaking incubator at 37°C, 8% CO2, and 125rpm for 24h. After 24h of culture, 10% Soytone was added to the culture medium to a final concentration of 0.5%, and the cells were further incubated at 37°C, 8% CO2, and 125rpm for an additional 120h. Six days post-transfection, the HEK293F cells were harvested by centrifugation at 3,800 rpm for 30min. The supernatant was collected for protein purification, and a primary purification step was performed using protein A affinity chromatography. The purity and molecular weight of the purified protein were analysed by SDS-PAGE and size-exclusion chromatography–high-performance liquid chromatography (SEC-HPLC). Human recombinant N-TSP1 protein (Ser24–Thr221) (RPA611Hu01, USCN) and recombinant mouse full-length TSP1 protein (F-TSP1; 7859-TH, R&D Systems) were purchased from a commercial protein supplier. PBS was used as the vehicle control for all in vitro and in vivo treatments.​