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. 2015 Jun 9;11(6):e1004960. doi: 10.1371/journal.ppat.1004960

Fig 1. TRIM32 interacts and translocates with influenza A virus PB1 protein.

Fig 1

(A) Primary human tracheal epithelial cells were infected with 0.1 MOI PR8 IAV for 16 hr. Whole cell lysates (WCL) were subjected to immunoprecipitation (IP) and immunoblotting with indicated antibodies to detect endogenous interactions. Molecular weights (MW) are indicated. (B) GST pull down of bacterially expressed GST-PB1 and HIS-TRIM32. (C) FLAG-tagged PB1 from 6 different influenza A strains [PR8, A/Puerto Rico/8/1934 (H1N1); WSN, A/WSN/1933 (H1N1); Aichi, A/Aichi/2/1968 H3N2; NY, A/New York/1682/2009 (H1N1); A/Vietnam/1194/2004 (H5N1); A/Anhui/1/2013 (H7N9)] were co-expressed with TRIM32-GFP in HEK293 cells. After 48 hr, cell lysates were immunoprecipitated with anti-FLAG and probed as indicated. As input controls, WCL were immunoblotted. (D) TRIM32 fused with HA epitope was co-transfected into HEK293 cells with PR8 derived FLAG-tagged PB1, PB2 or NP. After 48 hr, WCL were immunoprecipitated with anti-FLAG antibody and blotted with indicated reagents. TRIM32 can appear as a doublet on Western blots. (E) Primary human tracheal epithelial cells were infected with 0.01 MOI PR8 strain IAV for 8 or 16 hr and stained with anti-TRIM32 (green), anti-PB1 (red) and DAPI nuclear stain (blue). Right panel shows quantitated TRIM32-PB1 colocalization data. (F) A549 cells were infected with 0.01 MOI IAV PR8 strain for the indicated times, whole cell lysates (W) or cytosolic (C) and nuclear (N) fractions were extracted and blotted as indicated. Right panel depicts the densitometric ratio of nuclear to cytoplasmic PB1.