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. 2015 Jan 28;89(8):4058–4068. doi: 10.1128/JVI.03574-14

FIG 3.

FIG 3

C18 regulates B12, B14, and BAP31 focus formation. (A) RT-PCR results showing the knockdown efficiency of C18 (lanes 1 and 2)-, B12 (lanes 3 and 4)-, and B14 (lanes 5 and 6)-specific siRNAs. The expression of GAPDH mRNA was used as a loading control. (B) CV-1 cells were reverse transfected with scrambled or C18 siRNA. After 48 h of transfection, cells were infected with SV40 (MOI, ∼15) for 14 h, fixed, stained with the indicated antibodies, and analyzed by immunofluorescence microscopy. (C) Quantification data from panel B, where cells were scored positive if at least one focus was present in the cell. Values represent means ± SD from three independent experiments. (D) S/His-tagged Sel1L-transfected CV-1 cells were infected with SV40 (MOI, ∼50), fixed at 14 hpi, stained with anti-S and anti-BAP31 antibodies, and analyzed by immunofluorescence microscopy. (E) BAP31 interaction with C18, B14, and B12 were analyzed by using lysates derived from HEK 293T cells transfected with the indicated construct. The S-tagged proteins were affinity purified (AP) using S-agarose beads, and the precipitated samples were immunoblotted with the indicated antibodies. (F, G, and H) Interaction between C18-S/B14-S with endogenous B12 (F), C18-S/B12-S with endogenous B14 (G), and C18-S with endogenous Hsc70 and SGTA (H) were assessed by affinity purification using HEK 293T lysates harboring the indicated construct using S-agarose beads, followed by immunoblotting the precipitated material with the indicated antibodies. (I) S-tagged C18-expressing vector can rescue the C18 knockdown effect. CV-1 cells were reverse transfected with either scrambled or C18 siRNA (50 nM) for 24 h. Cells then were transfected with the indicated construct for 24 h, infected with SV40 (MOI, ∼0.5) for 20 h, fixed, and stained using anti-large T antigen, anti-FLAG, or anti-S antibodies. The percentages of large T antigen-positive cells were determined in cells expressing either GFP-FLAG or C18-S* by immunofluorescence microscopy. C18-S* is a construct designed to be resistant to the C18 siRNA. Values represent means ± SD from three independent experiments.