Fig. 7: Interaction between NRP1 and EGFR signaling in keratinocytes.

A Detection of the EGFR-ERK pathway and EMT inducers by western blotting in Hacat cells incubated with recombinant Sema3A and EGF for 48 h. B Protein levels of NRP1, EGFR, p-EGFR, ERK and p-ERK in cells transfected with si-NRP1. C Localization of NRP1 and EGFR proteins in Hacat cells. Scale bar = 20 µm. D Co-IP experiment between NRP1 and EGFR. IP: NRP1. WB: EGFR. E EGFR- and NRP1-overexpressing Hacat cells were treated with cycloheximide (CHX) for the indicated time periods to inhibit de novo protein synthesis. As a control, MG132 was added to block the catalytic activity. F si-NRP1 and EGFR plasmids were cotransfected into NHEK cells for 48 h. Protein levels of NRP1, EGFR, p-EGFR, ERK and p-ERK were determined by western blot. G NHEK cells were transfected with si-NRP1 plasmids for 2 days before EGF (50 ng/ml) stimulation. Then the IF analysis of EGFR or NRP1 was showed. Scale bar = 20 µm. H NHEKs were stimulated with EGF (100 ng/ml) for the indicated periods of time. IFs were subsequently conducted in the resulting cells to monitor EGFR and NRP1 localization/expression. Scale bar = 20 µm.