Fig. 2
SH3BGRL3 binds to the neck region of Myo1c in a calcium dependent manner. (a) Diagram of the wild type Myo1c domains and of the deletion mutants used for the Co-IP experiments. (b) SKBR3 cells were co-transfected with wild type and deletion mutant Myo1c constructs, each carrying an HA tag, and FLAG-SH3BGRL3 construct. FLAG-SH3BGRL3 and Myo1c variants were co-immunoprecipitated using anti-FLAG conjugated resin, and Myo1c variants revealed using an anti-HA antibody. Co-IP of a lysate from SKBR3 cells not transfected with any Myo1c construct was used as negative control. (c) Endogenous Myo1c was pulled-down using GST-SHBGRL3 fusion protein or GST alone bound to Sepharose-Glutathione 4B, in presence of Ca2+ or EGTA and revealed with an anti-Myo1c antibody. P-IQ: Post-IQ domain; PH: pleckstrin-homology domain; HA: HA tag