Overexpression of the LMAN1-MCFD2 complex does not further increase FV and FVIII secretion. (A) WT HepG2 and HepG2^LMAN1− cells with (+) or without (−) stably expressing LMAN1 were transfected with a MCFD2 expressing construct (+) or with an empty vector (−). Cell lysates were subjected to immunoblotting 48 hours after transfection with the indicated antibodies. (B) FV antigen levels were measured in conditioned media 48 hours after transfection. (C) WT 293T cells with (+) or without (−) stably overexpressing MCFD2 were cotransfected with a FVIII expression construct and a LMAN1 expressing construct (+) or an empty vector (−) as indicated. Cell lysates were subjected to immunoblotting 48 hours after transfection with the indicated antibodies. (D) FVIII activity and antigen levels were measured in conditioned media 48 hours after transfection. FV and FVIII level data presented are means of 3 independent experiments, and the error bars represent standard deviations.