DDOST potentiates the function of MITA at the ER.

(A, B) HeLa cells were transfected with Cherry-DDOST and GFP-MITA (A) or GFP-tagged organelle markers (B) and visualized under confocal microscopy 24 hours later. Vec: empty vector as a control; Scale bar: 10 μm. (C) DDOST knockdown HaCaT cells and control cells were infected with HSV-1 (MOI = 2) for 3 hours before staining with rabbit anti-MITA and Cy3 goat anti-rabbit IgG. Scale bar: 10 μm. (D) DDOST knockdown THP-1 cells and control cells were infected with HSV-1 (MOI = 2) for the indicated time. Cell lysates were subjected to Co-IP and immunoblotting analysis using the indicated antibody. (E) DDOST knockdown THP-1 cells and control cells were infected with HSV-1 (MOI = 2) for the indicated time before immunoblotting analysis. One representative result from at least three independent experiments is shown. (F, G) HEK293T cells were transfected with HA-MITA plus full length Flag-DDOST or its truncated mutants (F) or Flag-DDOST plus full length HA-MITA or its truncated mutants (G). Then, Co-IP and immunoblotting analysis were sequentially performed using the indicated antibodies 24 hours posttransfection. The related interaction diagrams are shown in the lower panels. FL: full length; TM: transmembrane domain; IgL: immunoglobin light chain.