Figure 1

Identification of CAD as an mLST8-binding protein. (A) Co-purification of CAD with FLAG-mLST8. The extract from HEK293 cells transfected with or without FLAG-mLST8 vector was subjected to immunoprecipitation with the anti-FLAG antibody, and the proteins eluted from resin with the FLAG peptide were visualized by silver staining after SDS-PAGE. FLAG-mLST8, mTOR, and the proteins recovered from the immunoprecipitate (No.1 to 4) are shown. The positions of the size standards are indicated in kDa. (B) Association of CAD with overexpressed mLST8. The extract from HEK293 cells transfected with the FLAG-mLST8 vector was subjected to immunoprecipitation with either the anti-CAD or anti-FLAG antibody, and the immunoprecipitated proteins were analyzed by immunoblot with the anti-CAD and anti-mLT8 antibodies. NRG (normal rabbit globulin) and NMG (normal mouse globulin) were employed as negative controls. *: a non-specific protein. (C) Association between myc-CAD and FLAG-mLST8. The extract from HEK293 cells transfected with myc-CAD and FLAG-mLST8 vectors was subjected to immunoprecipitation with either the anti-myc or anti-FLAG antibody, and the immunoprecipitated proteins were analyzed by immunoblot with antibodies. (D) Association between the endogenous CAD and mLST8. The HEK293 cell extract without transfection and the immunoprecipitate of the extract with the anti-mLST8 antibody were analyzed by immunoblot with the anti-CAD and anti-mLT8 antibodies. NRG was employed as a negative control.