Figure 5.
Overexpression of MCFD2 rescues FVIII secretion in LMAN1 KO and MCFD2 KO cells. (A) A FV or FVIII expression construct and vectors expressing the indicated MCFD2 variants were cotransfected into 293TMCFD2− cells in 24-well plates in 2 doses (50 ng and 20 ng). MCFD2 expression levels in cell lysates were compared with vector-transfected WT 293T and 293TMCFD2− cells by immunoblotting. (B) FV antigen levels in conditioned media were measured 48 hours after transfection. (C) FVIII activity and antigen levels in conditioned media were measured 48 hours after transfection. (D) A FV or FVIII expression construct and vectors expressing the indicated MCFD2 variants were cotransfected into 293TLMAN1− cells in 24-well plates in 2 doses (50 ng and 20 ng). MCFD2 expression levels in cell lysates were compared with vector-transfected WT 293T and 293TMCFD2− cells by immunoblotting. MCFD2 expression levels in cell lysates were compared with vector-transfected WT 293T and 293TLMAN1− cells by immunoblotting. (E) FV antigen levels in conditioned media were measured 48 hours after transfection. (F) FVIII activity and antigen levels in conditioned media were measured 48 hours after transfection. FVIII activity and antigen data presented are means of 3 independent experiments, and the error bars represent standard deviations. ∗∗P < .01; ∗∗∗P < .001.

Overexpression of MCFD2 rescues FVIII secretion in LMAN1 KO and MCFD2 KO cells. (A) A FV or FVIII expression construct and vectors expressing the indicated MCFD2 variants were cotransfected into 293TMCFD2− cells in 24-well plates in 2 doses (50 ng and 20 ng). MCFD2 expression levels in cell lysates were compared with vector-transfected WT 293T and 293TMCFD2− cells by immunoblotting. (B) FV antigen levels in conditioned media were measured 48 hours after transfection. (C) FVIII activity and antigen levels in conditioned media were measured 48 hours after transfection. (D) A FV or FVIII expression construct and vectors expressing the indicated MCFD2 variants were cotransfected into 293TLMAN1− cells in 24-well plates in 2 doses (50 ng and 20 ng). MCFD2 expression levels in cell lysates were compared with vector-transfected WT 293T and 293TMCFD2− cells by immunoblotting. MCFD2 expression levels in cell lysates were compared with vector-transfected WT 293T and 293TLMAN1− cells by immunoblotting. (E) FV antigen levels in conditioned media were measured 48 hours after transfection. (F) FVIII activity and antigen levels in conditioned media were measured 48 hours after transfection. FVIII activity and antigen data presented are means of 3 independent experiments, and the error bars represent standard deviations. ∗∗P < .01; ∗∗∗P < .001.

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