Binding
of inS3-54 to STAT3. (A) Ectopic overexpression of FLAG-STAT3
(S3) in H1299 cells. Vec = vector control. (B) Pull-down assay of
STAT3 from lysate of FLAG-STAT3-transfected H1299 cells using EAH-Sepharose
4B-conjugated without (vehicle control, VC) or with inS3-54 (inS3).
Pull-down materials were separated using SDS-PAGE and Western blot
analysis probed with STAT3 antibody (IB) or stained with silver (SS).
(C) Pull-down materials using EAH-Sepharose 4B-conjugated without
(VC) or with inS3-54 (inS3) were treated at room temperature (RT)
or by boiling before separation on SDS-PAGE for Western analysis.
(D) Competition of STAT3-binding to inS3-54-conjugated EAH-Sepharose
4B by excess free inS3-54 (inS3), an irrelevant compound (IC) control,
or vehicle control (VC). (E) Elution of STAT3 bound to inS3-54-conjugated
EAH-Sepharose 4B by inS3-54 (inS3), an irrelevant compound (IC) control,
or vehicle control (VC). (F) Pull-down assay of purified STAT3 with
input shown by silver staining (SS).