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. 2013 Aug 8;9(8):e1003533. doi: 10.1371/journal.ppat.1003533

Figure 2. Riplet and TRIM25 ubiquitin ligases associate with RIG-I.

Figure 2

(A, B) HeLa cells were transfected with Riplet-HA expression vector (A) or FLAG-Riplet and TRIM25-HA (B). 24 hours after transfection, the cells were infected with VSV at MOI = 1 for six hours. The cells were fixed and stained with anti-RIG-I (Alme-1), HA, and/or FLAG antibodies as indicated. Histograms display the measured fluorescence intensity along the white line in the merged panels. (C–F) Colocalization coefficients of Riplet localization to RIG-I (C) or TRIM25 (E) in mock or VSV infected HeLa cells. Pearson's correlation coefficient of Riplet and RIG-I (D) and TRIM25 (F) (mean ± SD, n>10). (G, H) HeLa cells were transfected with Riplet-HA (G) or TRIM25-HA (H) expression vector. The cells were stimulated with 100 ng of short polyI:C for six hours. The cells were fixed and stained with anti-G3BP and HA antibodies. Colocalization coefficient indicates values (mean ± SD, n>10) of Riplet (G) or TRIM25 (H) localization to G3BP staining region. Histograms display the measured fluorescence intensity along the white line in the merged panels. (I, J) TRIM25 (I) or Riplet (J) expression vector was transfected into HEK293FT cells together with FLAG-tagged-RIG-I CARDs or -RIG-I RD expression vectors. Cell lysate was prepared at 24 hours after transfection, followed by immunoprecipitation with an anti-FLAG antibody. (K) Riplet, RIG-I, and/or RIG-I-ΔRD, which lacks RD, expression vector was transfected into HEK293 cell with p125luc reporter. Reporter activation was determined at 24 hours after transfection. Data are presented as mean ± SD (n = 3).