Fig. 4
Elafin interacts with EGFR, promotes EGFR dimerization and internalization, and activates EGFR/AKT pathway. a Mass spectrometry analysis of Elafin-associated proteins in PLC-8024 cells. The list of peptide fragments (95% CI) and peptide coverage (95% CI) of membrane proteins is presented. b Endogenous Elafin interacted with EGFR in PLC-8024 cells. The whole-cell lysate and indicated concentrated condition medium were mixed and then immunoprecipitated. After separation on SDS gels, western blotting was conducted with the indicated antibodies. c Co-immunoprecipitated was performed to detect the interaction between Elafin and endogenous EGFR in Elafin overexpressing HCC cells. The whole-cell lysate was mixed up with concentrated vector or Elafin-overexpression condition medium and then were immunoprecipitated. d Schematic diagram of wild-type EGFR and its truncated mutants was shown. e Elafin could bind to the EGFR extracellular domain via subdomain I and III. HEK293T cells were transfected with indicated EGFR constructs and then lysed at 48-h. After incubation with the rElafin, the lysates were subjected to co-IP assays. f The putative binding mode of Elafin and EGFR. EGFR and Elafin were shown as light pink cartoon and teal cartoon, respectively. g Elafin bound to EGFR at the same binding site for EGF. EGFR, Elafin and EGF were shown as light pink cartoon, teal cartoon and yellow cartoon, respectively. h Induction of EGFR dimerization of Elafin. After starving and treatment with BSA as control, rElafin (10 μg/ml) or EGF (100 ng/ml), the cells were crosslinked with 3 mM BS3 and were subjected to western blotting assays. i rElafin promoted EGFR internalization. Huh7 cells were starved for 24 h and then treated with BSA, rElafin and EGF for 30 min, then immunofluorescence staining was performed. Scale bar, 20 μm. j Key members of EGFR downstream signaling expressions were detected by western blotting in Elafin knockdown and overexpressing HCC cells