P2138

To further explore the direct combination relationship of AjFGF4 and AjFGFR2, the pull‐down assay was performed as previously reported with minor modifications.²⁵ The E. coli Rosseta cells harboring induced expression of GST‐tag labeled AjFGF4 or GST‐tag were pelleted by centrifugation and lysed in lysis buffer via ultrasonication. After centrifugation at 13,000g at 4°C for 15 min, the GST‐tag and GST‐tag‐labeled AjFGF4 protein was purified by anti‐GST magnetic beads (P2138, Beyotime) and used for pull‐down assays. Subsequently, the GST magnetic beads were washed five times with lysis buffer to remove unbound proteins and were then used to incubate with HEK‐293T cell lysate overnight which was transfected with pcDNA‐flag‐FGFR2 plasmids. Finally, the beads were washed five times with 10 resin‐bed volumes of RIPA buffer for 10 min and then analyzed via immunoblot analysis to detect the flag‐FGFR2 proteins using an anti‐flag antibody (M20008, Abmart).

A GST pull‐down assay was performed using a Magne GST pull‐down system (Promega) according to the manufacturer's instructions. Briefly, proteins were expressed in Escherichia coli BL21 (DE3) overnight at 18°C, and purification of GST fusion proteins was performed with GST‐tag purification resin (P2253; Beyotime). Total cell lysates were subjected to GST pull‐down by magnetic beads overnight at 4 °C (P2138; Beyotime) with immobilized GST‐control or GST‐tagged proteins as indicated. The beads were then washed three times with chilled lysis buffer. Eluted samples and inputs were subjected to western blotting.