Panel A shows a schematic diagram summarizing Endophilin A1 and Endophilin A2 domain constructs with BAR and SH3 deletions. Panel B shows Western blot images demonstrating ALFA-tag co-immunoprecipitation of EGFP-Endophilin-A1 with ELKS and CAST proteins. Panel C shows Western blot images demonstrating ALFA-tag co-immunoprecipitation of EGFP-Endophilin-A2 with ELKS and CAST proteins. Panel D shows Western blot images comparing interactions of Endophilin A1 BAR and SH3 domain constructs with ELKS. Panel E shows Western blot images comparing interactions of Endophilin A2 BAR and SH3 domain constructs with CAST after ALFA-tag precipitation.
Physiological interaction of CAST/ELKS and endophilin-A in the HEK cells. (A) Cartoon image of the deletion constructs of endophilin-A1 and endophilin-A2. (B and C) Binding of CAST or ELKS to endophilin-A1 (A), or endophilin-A2 (B). HEK cells expressing EGFP-endophilin-A1 were precipitated with ALFA-mCherry, ALFA-mCherry-ELKS, or ALFA-mCherry-CAST with recombinant ALFA nanobody-GST protein-binding GSH-Sepharose. Precipitated EGFP–endophilin-A1 or EGFP–endophilin-A2 was detected by western blot analysis. (D and E) BAR domain of endophilin-A1– (D) or endophilin-A2– (E) mediated binding of CAST. HEK cells expressing EGFP–endophilin-A1-BAR or EGFP–endophilin-A1-SH3, or EGFP–endophilin-A2-BAR or EGFP–endophilin-A2-SH3 was precipitated with ALFA-mCherry, ALFA-mCherry-ELKS, or ALFA-mCherry-CAST with recombinant ALFA nanobody-GST protein-binding GSH-Sepharose. Precipitated EGF–endophilin-A1 or EGFP–endophilin-A2 were detected by western blot analysis. Source data are available for this figure: SourceData FS2.