Fig. 5: GREM1 stimulates EGFR signalling.

a–c Effect of GREM1 on EGFR signalling. a SKBR3 cells were starved overnight and stimulated with EGF or GREM1 (50 ng/ml, each) for 5, 15 or 30 min. The lysates were then immunoblotted with the indicated antibodies. b SKBR3 cells were stimulated with different concentrations of recombinant GREM1 protein (10, 50 or 100 ng/ml) for 15 min. EGF (50 ng/ml) was used as a positive control, and the lysates were collected for immunoblot analysis. c Immunoblot assay for detection of EGFR and related signalling molecules in breast cancer cells overexpressing GREM1. Protein lysates isolated from each indicated cell were subjected to immunoblot analysis as described in ‘Methods’ section. d Effect of erlotinib on EGFR signalling induced by GREM1. SKBR3 cells were incubated with recombinant GREM1 protein in the absence or presence of erlotinib (1 μM) for 15 min and the lysates were immunoblotted with the indicated antibodies. e Flow cytometric analysis of interaction between EGFR and Fc-GREM1. HEK293 cells overexpressing Flag-only, Flag-EGFR or Flag-BMP2 were incubated with either Fc-ctrl (Fc-IgG1) or Fc-GREM1 protein for 1 h at 4 °C, followed by further incubation with Alexa Fluor 647 goat anti-human IgG (H + L) antibody for 30 min. Flow cytometric analysis was carried out using a FACS Canto II instrument. f HEK293 cells overexpressing Flag-only, Flag-EGFR or Flag-BMP2 were incubated with either Fc-ctrl (Fc-IgG1) or Fc-GREM1 protein for 1 h at 37 °C, and the lysates were subjected to immunoprecipitation using anti-Flag antibody. Immune complexes were immunoblotted with anti-Fc antibody.