Active 3C protease is sufficient to degrade nuclear proteins/nucleoporins in transfected cells.

<p>(<b>Ai</b>) COS-7 cells transfected to express either GFP-3C or GFP-3Cinac were trypsinized 18 h after transfection, harvested in ice-cold PBS and FACS sorted to collect GFP-expressing cells. Cells were then lysed using RIPA buffer plus protease and phosphatase inhibitors and lysates were subjected to Western analysis as per <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071316#pone-0071316-g001" target="_blank">Figure 1A</a>; untransfected cells were lysed similarly and used as control. The antibodies used are shown on the left of the figure. The arrow in the nucleolin blot denotes a clear cleavage product, with the approximate molecular weight (kDa) indicated on the right. (<b>Aii</b>) Results for densitometric analysis of nucleolin and Nup153 protein bands such as those shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071316#pone-0071316-g003" target="_blank">Figure 3A</a>i, where data were normalised to the corresponding value of tubulin, relative to the corresponding value for the control sample. Densitometric analyses were performed using ImageJ; values represent the mean (+ SD) from two independent experiments (<b>Bi</b>) Ohio-Hela whole cell lysates were incubated with 4 units of HRV14 3C protease at 37°C for the indicated incubation times, subsequent to SDS-PAGE on 10% gels and Western analysis as per <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071316#pone-0071316-g001" target="_blank">Figure 1A</a>. (<b>Bii</b>) Plot of densitometric analysis of protein bands in (Bi), where data were normalised to the corresponding values for tubulin, relative to 0 h samples. Densitometric analyses were performed using Image J and values were the mean of two different experiments (± SD). (<b>C</b>) COS-7 cells transfected to express either GFP-3C or GFP-3Cinac were fixed and permeabilized 18 h post-transfection, and immunostained as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071316#pone-0071316-g002" target="_blank">Figure 2</a> with the indicated primary and Alexa-568 conjugated secondary antibodies. Fluorescence was imaged by CLSM (see Materials and Methods). In each panel, images on the left depict localisation of HRV16 proteins (green channel) and the images in the middle depict localisation of cellular proteins (red channel), with the merged image on the right.