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. 2014 Dec 16;89(1):300–311. doi: 10.1128/JVI.02170-14

FIG 5.

FIG 5

AIMP2 does not alter the subcellular distribution of NS2 but enhances the stability of M1. (A) AIMP2 does not alter the cellular localization of NS2. 293T cells were cotransfected with Flag-AIMP2 and HA-NS2. At 30 h posttransfection, cytoplasmic and nuclear proteins were extracted and assayed by Western blotting with the indicated antibodies. Tubulin and lamin B1 were used as loading controls for the cytoplasmic and nuclear fractions, respectively. Relative NS2 band intensities were measured using ImageJ software (NIH). Data represent the mean ± SD from three independent experiments. One-way ANOVA was used to determine the differences between independent groups. The differences between the variants were considered statistically significant if P was <0.05. (B) Knockdown of AIMP2 decreases the nuclear import and stability of M1. 293T cells were transfected with siCtr or siAIMP2 and infected with the A/WSN/33 virus at an MOI of 2. At 8 and 16 h p.i., cytoplasmic and nuclear proteins were extracted and assayed by Western blotting with the indicated antibodies. Data are representative of those from three independent experiments. (C) Quantification of relative NP, M1, and NS2 band intensities from panel B using ImageJ software (NIH). Data represent the mean ± SD from three independent experiments. Statistical significance was determined by ANOVA. (D) Enhancement of M1 nuclear localization in AIMP2-overexpressing cells. A549 and 293T cells were transfected with HA-AIMP2 and infected with A/WSN/33 at an MOI of 2. At 6 h p.i., the cells were fixed, permeabilized, and stained for HA-AIMP2 and M1. White arrows, AIMP2-overexpressing cells; yellow arrows, AIMP2-nonoverexpressing cells. Data are representative of those from three independent experiments. (E) 293T cells were transfected with siCtr, siAIMP2, or siCtr plus pcDNA-AIMP2 for 48 h, followed by infection with the A/WSN/33 virus at an MOI of 2 for 8 h. Whole-cell lysates were assayed by Western blotting with the indicated antibodies. Relative band intensities were measured using ImageJ software (NIH). Data represent the mean ± SD from three independent experiments. Statistical significance was determined by ANOVA. ***, P < 0.001; **, P < 0.01; *, P < 0.05; ns, no significant difference.