Fig 2: The NPC subunit NUP188 binds to Nesprin-2 and promotes SV40 nuclear entry.(A) CV-1 cells were infected with SV40 (MOI ~1) and treated with either a dimethyl sulfoxide (DMSO) control or importazole. At 24 hpi, cells were fixed and stained for large TAg. Data were normalized to the DMSO control. (B) Unique peptides corresponding to SV40-interacting host proteins identified by IP mass spectrometry [32]. (C) CV-1 cells were transfected with scrambled control siRNA, 50 nM siRNA against NUP35, NUP88, NUP93, NUP98, and NUP133, or 10 nM siRNA against NUP188 for 48 h. Protein levels were assessed by immunoblotting. Hsp90 was used as a loading control. (D) CV-1 cells were transfected as in (C) and infected with SV40 (MOI ~1) for 24 h. Cells were fixed and stained for large TAg. Data were normalized to the scrambled control. (E) CV-1 cells were infected with SV40 (MOI ~25) for 20 h. Endogenous NUP188 was immunoprecipitated (IP) from whole-cell extracts and the eluted samples subjected to SDS-PAGE followed by immunoblotting for VP1. (F) CV-1 cells were infected with SV40 (MOI ~25) for 20 h. Endogenous Nesprin-2 was immunoprecipitated (IP) from whole-cell extracts and eluted samples subjected to SDS-PAGE followed by immunoblotting for NUP188 and BICD2. (G) CV-1 cells were transfected with either the scrambled control siRNA or siRNA against Nesprin-2 for 48 h. Cells were then infected with SV40 (MOI ~25) for 20 h and endogenous NUP188 immunoprecipitated from whole-cell extracts. The samples were subjected to SDS-PAGE followed by immunoblotting for VP1. (H) As in (G), except samples were immunoblotted for NUP93. (I) Confocal analysis of CV-1 cells that were transfected with either scramble control siRNA or siRNA against NUP188 for 48 h and then infected with SV40 (MOI ~25) for 20 h in the presence of 1 μM actinomycin D (ActD). Cells were stained with anti-VP2/3 (green) and anti-MAB414 that stains the NPC (red). Cells were counterstained with DAPI (blue). White arrow = VP2/3 at nuclear membrane, orange arrow = VP3 inside the nucleus. (J) Quantification of the percentage of cells in (I) with VP2/3 at the nuclear membrane normalized to the scrambled control. Actual values are shown in parenthesis. (K) As in J, except the percentage of cells with VP3 inside of the nucleus is quantified. Actual values are shown in parenthesis. Values are averages of the means (n = 3) ± SD. A standard Student’s t test was used to determine statistical significance. **, P ≤ 0.005; ***, P ≤ 0.0005; ****, P ≤ 0.0001. Scale bars, 10 μm.