Figure 5. GPS2 is indispensable for the interaction between NS5A and VAP-A.

(A) Overexpression of GPS2 enhances the interaction between NS5A and VAP-A. 293T cells (2×10⁶) were transfected with the indicated expression plasmids (6 µg each). Cell lysates were immunoprecipitated with anti-Flag (F) antibody or control mouse lgG (Ig). The immunoprecipitates were analyzed by Western blots with anti-HA antibody (upper panel). The expression levels of transfected proteins were analyzed by Western blots with anti-Flag or anti-HA antibody (Lower panel). (B) Knockdown of GPS2 disrupts the interaction between NS5A and VAP-A in 293T cells. 293T cells (2×10⁶) were transfected with the indicated plasmids. Cell lysates were immunoprecipitated with anti-Flag (F) antibody or control mouse lgG (Ig). The immunoprecipitates were analyzed by Western blots with anti-HA antibody, while the expression levels of proteins in cell lysates were analyzed by Western blots with indicated antibody. (C) Knockdown of GPS2 disrupts the interaction between NS5A and VAP-A in hepatoma cells. sh-GPS2 or sh-NT transduced stable Huh7 cell lines were transfected with the indicated plasmids. Cell lysates were immunoprecipitated with anti-Flag (F) antibody or control mouse lgG (Ig). The immunoprecipitates were analyzed by Western blots with anti-HA antibody, while the expression levels of proteins in cell lysates were analyzed by Western blots with indicated antibody. (D) Neither overexpression nor knockdown of GPS2 influences VAP-A/B expression. Huh7 cells were transiently transfected with Flag-GPS2 or control vector. VAP-A/B mRNA levels were determined by qRT-PCR 48 h later in these cells. The amount of VAP-A/B mRNA to that of GAPDH were calculated and graphed. In another assay, VAP-A/B mRNA levels in Huh7 cells with stable knockdown of GPS2 expression and control cells were determined by real-time qRT-PCR.