Fig. 2.

Interaction between Fhod3 and cMyBP-C. (A and B) Proteins in lysates of hearts from transgenic mice expressing Fhod3CM-WT were immunoprecipitated with the anti–Fhod3-C-20 (A) or the anti–cMyBP-C (B) antibodies, and then analyzed by immunoblot with the indicated antibodies. Uncropped images are shown in SI Appendix, Fig. S5. (C) Proteins in lysates of hearts from nontransgenic C57BL/6 mice were immunoprecipitated with the anti–Fhod3-C-20 or control IgG, and then analyzed by immunoblot with the anti–cMyBP-C antibody. Uncropped images are shown in SI Appendix, Fig. S5. (D) Schematic structures of various Fhod3 constructs used in the present pull-down binding assay (E–K). (E) FLAG-tagged Fhod3CM(FL) or Fhod3CM-S(FL) in the lysate of HEK-293F cells (input) was incubated with GST–cMyBP-C (C0C1 or C0C2) or GST alone and pulled down with glutathione-Sepharose 4B beads. Precipitated proteins were subjected to SDS/PAGE and analyzed by immunoblot with the anti-FLAG antibody (Upper) or stained with Coomassie Brilliant Blue (CBB) (Lower). Positions for marker proteins are indicated in kilodaltons. (F) FLAG-tagged full-length (FL) or N terminus (N) of Fhod3CM in the lysate of HEK-293F cells (input) was incubated with GST–cMyBP-C (C0C1 or C0C2) or GST alone and pulled down with glutathione-Sepharose 4B beads. Precipitated proteins were analyzed as in E. (G) Purified N-terminal region of Fhod3 proteins [Fhod3CM(N) or Fhod3CM-S(N)] tagged with FLAG was incubated with GST–cMyBP-C (C0C1 or C0C2) or GST alone and pulled down with glutathione-Sepharose 4B beads. Precipitated proteins were analyzed as in E. (H and J) Schematic structures of various cMyBP-C constructs used in I and J, respectively. (I and K) FLAG-tagged Fhod3CM(N) in the lysate of HEK-293F cells was incubated with indicated GST-fused fragments of cMyBP-C and pulled down with glutathione-Sepharose 4B beads. Precipitated proteins were analyzed as in E. The arrowheads indicate the position of Fhod3CM(N).