Fig. 1: TMEM25 interacts with EGFR.

a, b TMEM25 interacts with EGFR endogenously. Cell lysates from MDA-MB-231 cells were subjected to anti-TMEM25 IP followed by immunoblotting to detect associated EGFR (a) or to anti-EGFR IP followed by immunoblotting to detect associated TMEM25 (b). c TMEM25 colocalizes with EGFR at plasma membrane. MDA-MB-231 cells transfected with C-terminal green fluorescent protein (GFP)-tagged TMEM25 (TMEM25/GFP) were subjected to immunofluorescence assay to detect localization of TMEM25/GFP (green) and endogenous EGFR (red). The nuclei were stained with DAPI (blue). The scale bars indicate 10 μm. d TMEM25 interacts with EGFR via both extracellular domain and cytosolic domain. HEK293T cells transfected with indicated combinations of full length (F), N-terminal extracellular/transmembrane domains (N), or cytosolic domain (C) of TMEM25/GFP and C-terminal Flag-tagged EGFR (EGFR/F) were subjected to anti-Flag IP followed by immunoblotting assay to detect associated TMEM25. e Direct interaction between cytosolic domains of TMEM25 and EGFR. Bacterially expressed and purified His-tagged TMEM25 cytosolic domain (His/TMEM25-C) and GST-tagged EGFR cytosolic domain (GST/EGFR-C) were subjected to GST pull-down assay. f TMEM25 interacts with EGFR but not HER2, HER3, or HER4. HEK293T cells transfected with indicated combinations of C-terminal hemagglutinin (HA)-tagged EGFR, HER2, HER3, or HER4 (HERs/HA) and C-terminal Flag-tagged TMEM25 (TMEM25/F) were subjected to anti-Flag IP followed by immunoblotting assay to detect associated HERs. The blotting experiments are representative of at least three biologically independent replicates (a, b, d–f). Source data are provided as a Source Data file.