Analysis of Ad3 Pb mutants.

<p>(<b>A</b>) Sequence of Ad3 Pb N-terminus. The first PPxY motif is underlain in grey, the second PPxY motif is written in bold italics. The first amino acid residue of deletants is written in bold and underlined. (<b>B</b>) Dd production in the baculovirus system. Cell lysates were purified by centrifugation on a sucrose density gradient. Fractions recovered from 15–25% sucrose (Pb) and from 30–40% sucrose (Dd) were pooled separately, run on SDS-PAGE and analyzed by gel densitometry. (<b>C</b>) Relative production of Dd and Pb by N-terminal deletants. Data for Dd and free Pb production are expressed as a percentage of total Pb protein expression. The average from four electrophoretic runs is shown. (<b>D</b>) Native agarose gel electrophoresis (CBB stain) of proteins recovered from the Q-Sepharose column at a salt concentration corresponding to the elution of Dd. The fractions with the highest amount of protein were used. (<b>E</b>) Internalization of Dd mutants. Q-Sepharose-purified proteins were applied onto HeLa cells and intracellular Dd was visualized by Western blot or by flow cytometry (<b>F</b>) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046075#s4" target="_blank">Materials and Methods</a>.