Extended Data Fig. 2: FBXL7 mediates the degradation of active c-SRC.
a, HEK-293T cells were transfected with the indicated FLAG-tagged F-box proteins (FBPs) or an empty vector (EV). Twenty-four hours after transfection, cells were harvested and lysed. Whole-cell extracts (WCE) were subjected to immunoprecipitation (IP) with an anti-FLAG resin and immunoblotting. b, PNT1A cells were treated with MG132 during the last 3 h before lysis. Lysates were immunoprecipitated with either an antibody against c-SRC, an antibody to FBXL7, or nonspecific IgG, and immunoblotted. c, HEK-293T cells were transfected with FLAG-tagged wild-type FBXL7, FLAG-tagged FBXL7(ΔF), or an empty vector (EV). Twenty-four hours after transfection, whole-cell extracts (WCE) were subjected to immunoprecipitation (IP) with an anti-FLAG resin and immunoblotting. d, HEK-293T cells were transfected with FLAG-tagged wild-type c-SRC, FLAG-tagged c-SRC(Y530F), FLAG-tagged c-SRC(Y419F), or an empty vector (EV). Twenty-four hours after transfection, whole-cell extracts (WCE) were subjected to immunoprecipitation (IP) with an anti-FLAG resin and immunoblotting. e, DU145 cells were transfected with a pool of four siRNAs to FBXL7 or a non-targeting (NT) siRNA oligo for 48 h, and treated with cycloheximide (CHX) for the indicated times. Protein extracts were then immunoblotted as indicated. a–e, two independent experiments were performed with similar results.
