Characterization of the interactions of various TRIM56 mutants with TRIF.A, co-immunoprecipitation (co-IP) analysis of the association of Flag-TRIFSA and indicated TRIM56-V5 WT/mutant in cotransfected HEK293 cells. Cell lysates were immunoprecipitated (IP) with anti-V5, followed by immunoblotting (IB) with anti-Flag or anti-V5 (lanes 1–4). Immunoblotting of the expression of Flag-TRIFSA and TRIM56-V5 (WT or indicated mutant) in cell lysates are shown in lanes 5-8. Note that TRIFSA (TRIF lacking the C-terminal RHIM motif) was used to avoid the strong apoptotic effect of WT TRIF. B, Co-IP in HEK293-FIT-T56 WT/mut cells co-expressing Flag-TRIFSA and WT or indicated mutant HA-TRIM56 (induced by addition of Tet (+Tet)). Cell lysates were IP with anti-TRIM56, followed by IB with anti-Flag or anti-HA. The bottom blot shows expression of HA-TRIM56 or the indicated mutant and Flag-TRIFSA in cell lysates. Actin serves as loading controls. TRIM56, tripartite-motif protein-56.