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. 2016 Apr 14;90(9):4658–4669. doi: 10.1128/JVI.02964-15

FIG 8.

FIG 8

NP1 increased VP mRNA production independently of RNA splicing at the A3 splice acceptor. (A) HBoV1 intron deletion/exchange constructs. Plasmids pCMVNS*(In3Δ)Cap, pCMVEpoIn14(In3Δ)Cap, and pCMVEpoIn124Cap are diagrammed, with replaced introns shown. (B) Western blot analysis of capsid proteins. HEK 293 cells were transfected with plasmids, as indicated, with (+) or without (−) cotransfection of pOpt-NP1. The cells were harvested and lysed 2 days posttransfection. The lysates were analyzed by Western blotting using an anti-VP antibody and reprobed with anti-β-actin. The lysates were also analyzed by Western blotting using anti-NP1 and anti-HA (for mCherry) antibodies. (C) Northern blot analysis of VP mRNAs. HEK 293 cells were transfected with plasmids as indicated. The cells were harvested, and total RNA was extracted 2 days posttransfection. The total-RNA samples were analyzed by Northern blotting using the NSCap probe. EB-stained 18S rRNA bands are shown. Detected bands of VP mRNA and (pA)p mRNA are indicated. The asterisk denotes various NS-encoding mRNAs. Lane M, RNA ladder marker. (D) Quantification of VP mRNAs on a Northern blot. The bands of VP mRNA and (pA)p mRNA in each lane of panel C were quantified and normalized to the level of 18S rRNA. The intensity of the VP mRNA band in lane 4 was arbitrarily set as 100%. Relative intensities were calculated for the bands in the other lanes. Means and standard deviations were calculated from the results of three independent experiments. The P values shown were calculated using a two-tailed Student t test. **, P < 0.01. (E) Determination of the usage of the A1, D2, and A3 splice sites using RPA. Ten micrograms of total RNA isolated 2 days posttransfection from HEK 293 cells transfected with plasmids as indicated was protected by the pA1D2 and pA3 probes or by their homology counterparts, as indicated. Lane M, 32P-labeled RNA markers (22), with sizes indicated on the left. The origins of the protected bands are shown with sizes. Spl, spliced RNAs; Unspl, unspliced RNAs.