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. 2023 May 30;19(5):e1011383. doi: 10.1371/journal.ppat.1011383

Fig 3. Interaction and colocalization of IFITM1 and AiV proteins, 2B, 2BC, 2C, 3A, and 3AB.

Fig 3

(A) A mammalian two-hybrid assay was performed by transfecting Vero cells with the indicated combinations of pACT and pBIND plasmids along with the pG5luc firefly luciferase reporter plasmid. At 48 h after transfection, the normalized firefly luciferase activity (firefly luciferase activity/Renilla luciferase activity) was measured and represented as fold of the control activity, which was obtained by combination with the empty pACT or empty pBIND plasmid. Data are the mean ± SD of at least three independent experiments. (B) 293T cells were transfected for 24 h with FLAG-tagged IFITM1 and HA-tagged 3Cm, 2B, 2BC, 2C, 3A, or 3AB, or HA-tagged IFITM1 and FLAG-tagged TGN46 expression plasmids, as indicated, followed by coimmunoprecipitation (IP) with an anti-FLAG, -HA or control IgG antibody. The resulting immunoprecipitates and whole-cell lysates were subjected to immunoblotting (IB) with anti-FLAG and anti-HA antibodies. (C) Vero cells were transfected with FLAG-IFITM1. At 24 h after post-transfection, the cells were fixed and double stained with anti-IFITM and anti-EEA1, anti-CD63, or anti-LBPA antibodies, as indicated. Pearson correlation coefficient analyses for data were obtained from ≥10 cells. Correlation coefficients are presented as the mean and standard deviation. (D) Vero cells was transfected with FLAG-IFITM1 and HA-2B, HA-2BC, HA-2C, HA-3A, or HA-3AB. At 24 h after transfection, cells were fixed and stained with anti-FLAG and anti-HA antibodies. Bars, 4 μm. Pearson correlation coefficient analyses for data were obtained from 4–8 cells.