Figure 4.

(A) HEK293T cells were transfected with HA-Src constructs for 24 h and treated with Pg-EE as indicated. Western blot analysis was performed for HA, total Src, and phosphorylated Src (Tyr416). (B–E) After overexpressing the Src and Src domain deletion constructs in HEK293T cells, a cellular thermal shift assay (CETSA) was performed with Pg-EE (150 µg/mL) or DMSO (as a control). Stabilization of Pg-EE on Src was examined by western blot analysis. The calculation of band intensity of Src in Pg-EE- and DMSO-treated groups was performed with Image J. (F) RAW264.7 cells (5 × 10⁶ cells/mL) were incubated with Pg-EE in the presence or absence of LPS (1 µg/mL) for 0 or 5 min. The binding of phospho-p85 (Tyr458) to Src was detected in whole cell lysates by immunoprecipitation with antibodies to Src and immunoblotting with antibodies to p-p85. −: no treatment and +: treatment.