Identification of EBRs within the Ft ICD. (A) Identification of EBR1 in the Ft ICD. HEK293 cell expression and IP of indicated FLAG-tagged Ft^ΔECD constructs in the presence of Ex^FERM. (B) Identification of EBR2 in the Ft ICD. HEK293 cell expression and IP of indicated FLAG-tagged Ft^ΔECD or FLAG- and myristoyl-tagged constructs in the presence of Ex^FERM. (C) EBR2 directly binds Ex^NT. In vitro transcribed and translated Ex^NT was incubated alone or with biotin-tagged EBR2^WT or EBR2^MUT peptide (sequences indicated—mutant peptide containing six alanine substitutions) and subjected to streptavidin-purification. EBR2 sequence defined by co-IP and the conserved E region (highlighted in red) are also indicated. (D) Identification of the Ex interacting region of the Ft ICD. HEK293 cell expression and IP of indicated FLAG-tagged Ft^ΔECD constructs in the presence of Ex^FERM compared to FLAG-bead controls. The expression and presence of proteins was analyzed by immunoblotting with the indicated antibodies. Ft presents as multiple bands due to proteolytic processing (Feng and Irvine, 2009; Sopko et al., 2009). n/a indicates no Flag-tagged protein added. (E) Graphical scheme highlighting the Ft constructs used in the figure. In addition, the transmembrane domain (TM), EBR1, EBR2, and established conserved and function domains of the Ft ICD are depicted. In binding column: “++” denotes constructs that interact strongly to Ex^FERM and “−” denotes no interaction with Ex^FERM. WB, Western blot. Source data are available for this figure: SourceData F3.