Propeptide sequence controls the Ad5 precursor pVII protein stability.

<p>(<b>A</b>) Codon optimization of pVII sequence enhances the protein detection. HEK293T cells were transiently transfected with two different concentrations, 100ng (100) and 300ng (300), of the original Ad5 pVII(wt)Flag, codon-optimized (Codon opt.) Ad5 pVII(wt)Flag and Ad5 pVII(Δ24)Flag expressing plasmids. Protein expression was detected by Western blotting using anti-Flag and anti-Actin antibodies. (<b>B</b>) Decay of the pVII(wt)Flag protein is faster compared to the pVII(Δ24) protein. Doxcycline (Dox)-inducible HEK293 stable cell lines, expressing the pVII(wt)Flag and pVII(Δ24)Flag proteins for 12 hours, were treated with cycloheximide (CHX) (100µg/ml) for defined time points (1, 2, 3, 6 and 9 hours). Protein accumulation was quantitated by Western blotting and the Flag-tagged protein levels were normalized to Actin protein levels. Average from three independent experiments is shown. (<b>C</b>) Inhibition of proteasome enhances pVII(wt) detection. HEK293 stable cell line, expressing the pVII(wt)Flag protein after 12 hours of induction with Dox was treated with CHX (100µg/ml) for 3 hours (lanes 3 and 4). To block proteasomal activity the cells were simultaneously treated with MG132 (25μM) for 3 hours (lane 4). Protein expression was visualized by Western blotting using anti-Flag, anti-c-Myc and anti-Actin antibodies. Asterisk (*) indicates the detection of the unspecific proteins by the antibodies. Black arrow points to the migration of the c-Myc protein. Hyphen (-) indicates non-transfected or non-Dox induced cells.