STING Binding Interaction with PC7A Copolymer

To investigate the STING interaction with PC7A copolymer, we labeled the PC7A copolymer with biotin (2–3 biotins per polymer chain). For THP-1 cell pulldown assay, PC7A-biotin (200 μg/ml) was incubated with THP-1 cells for 8 hrs, and then cells were collected and lysed in RIPA buffer (Sigma R0278). Lysates were precipitated with streptavidin-modified dynabeads (BD 557812). Samples were analyzed using SDS-PAGE and Western blots by rabbit anti-STING antibody (Cell Signaling, Cat # 13647). For STING protein pull down assay, human STING CTD (139–379) expression and purification had been described before^{39 (link)}, PC7A-biotin(50 μg/mL) was incubated with STING CTD(1 μg/ml) for 3hrs in PBS (pH=6.8), PEPA-biotin in PBS (pH=6.8) and PD5A-biotin in PBS (pH=4.4) with the same concentration were used as control groups, and then precipitated with streptavidin-modified dynabeads. Samples were analyzed using SDS-PAGE and Western blots.