Fig. 4.

FBXO7 mediates Lys-63-linked poly-ubiquitination of NRAGE without affecting NRAGE levels. a HEK293 cells were transfected with increasing amounts of Flag-FBXO7. Endogenous NRAGE expression was confirmed by immunoblotting with the anti-NRAGE antibody. To determine equal loading, cell lysates were analyzed by immunoblotting with the anti-tubulin antibody. b, c HEK293 cells were transfected for 24 h with Myc-NRAGE alone or in combination with Flag-FBXO7. Cell lysates were immunoprecipitated with the anti-Myc antibody and immunoblotted with anti-K63 or anti-K48-ubiquitin antibodies. Ubiquitin, FBXO7, or NRAGE expression in cell extracts was determined by immunoblotting with anti-K63, anti-K48-ubiquitin, anti-Myc, or anti-Flag antibodies. d HEK293 cells were transfected for 24 h with plasmids encoding HA-tagged K63-ubiquitin plus Myc-tagged NRAGE alone or together with Flag-tagged FBXO7. Co-IP experiments were performed as described