Rescue of FV and FVIII secretion in LMAN1 KO cells by LMAN1 variants. (A) WT 293T cells were transfected with WT LMAN1 or the vector. 293T^LMAN1− cells were transfected with WT LMAN1, the indicated LMAN1 variants or the vector. Forty-eight hours after transfection, conditioned media were collected and cell lysates were subjected to immunoblotting analyses with anti-LMAN1, anti-MCFD2, and anti–β-actin antibodies. FVIII activity (B) and antigen (C) levels in conditioned media were measured. (D) WT LMAN1 and the indicated LMAN1 variants were stably expressed into HepG2^LMAN1− cells and LMAN1 expression levels were compared with vector-transduced WT HepG2 and HepG2^LMAN1− cells by immunoblotting. (E-F) A FVIII expression construct was transfected into HepG2^LMAN1− cells stably expressing indicated LMAN1 variants and conditioned media were collected 48 hours after transfection. FVIII activity and antigen levels in conditioned media were measured. (G) Conditioned media were collected 48 hours after a fresh medium change. FV antigen levels were measured by ELISA. FV and FVIII level data presented in this figure are means of 3 independent experiments, and the error bars represent standard deviations. ∗∗P < .01; ∗∗∗P < .001. ns, not significant.