Rescue of FV and FVIII secretion in MCFD2 KO cells by MCFD2 variants. (A) WT MCFD2 and the indicated MCFD2 variants were stably expressed in HepG2^MCFD2− cells and MCFD2 expression levels were compared with vector-transduced WT HepG2 and HepG2^MCFD2− cells by immunoblotting. A FVIII expression construct was transfected into these cells and conditioned media were collected 48 hours after transfection. FVIII activity (B) and antigen (C) levels in conditioned media were measured. (D) Conditioned media were collected 48 hours after a fresh medium change. FV antigen levels were measured by ELISA. (E) WT MCFD2 and the indicated MCFD2 variants were stably expressed in 293T^MCFD2− cells and MCFD2 expression levels were compared with vector-transduced WT 293T and 293T^MCFD2− cells by immunoblotting. A FVIII expression construct or a FV expression construct was transfected into these cells and conditioned media were collected 48 hours after transfection. FVIII activity (F), FVIII antigen (G), and FV antigen (H) levels in conditioned media were measured. FV and FVIII level data presented in this figure are means of 3 independent experiments, and the error bars represent standard deviations. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001.