Fig. 3. Effects of MG132 and leupeptin on ubiquitylation of Flag-NPC1. COS cells were transfected with myc/His6-ubiquitin (myc/His6-Ubi) and Flag-NPC1 constructs, and were cultured in the indicated medium for 6 hours in the absence or presence of MG132 (upper panel) or leupeptin (lower panel); the arrowhead indicates the top of the separating gel. Anti-Flag immunoprecipitation (IP) products were subjected to immunoblotting (IB) with anti-Flag or anti-myc. All results shown are representative and were reproduced at least twice.