HIV-1 gp160 traffics to the Golgi network in Ubc9 knockdown cells.
2013-07-08T02:06:47Z (GMT) by
<p>(a) Env trafficking through the secretory pathway. 293T cells were transfected with pNL4-3 alone, or in combination with either Ctr. siRNA or Ubc9 siRNA, or left untransfected (Un). Unlike the previous and subsequent experiments, cells were pulse (P) labeled with [<sup>35</sup>S] methionine/cysteine for a shorter period of time (30 minutes) and chased for 2 and 4 hours. Cell and media associated viral proteins were solublized and immunoprecipitated with pooled AIDS patient sera, split equally, and incubated for 3.5 hours at 37°C in the presence, or absence of Endo H <sub>f</sub>. Samples were separated by SDS PAGE and visualized by phosphorimaging using The Discovery Series Quantity One software. A representative, over-exposed gel is shown so that partially Endo H<sub>f</sub> resistant Env can be more easily visualized. Viral proteins and their positions in the gel are labeled on the left. The identity of Endo H<sub>f</sub>, untreated viral proteins and their positions in the gel are labeled on the right. Deglycosylated Endo H<sub>f</sub> sensitive forms of gp160 residing in the ER are labeled as gp160s. Partially deglycosylated, Endo H<sub>f</sub> resistant forms of gp160 and gp120 that have undergone glycan modification in the TGN are labeled as gp160r and gp120r. gp160r in Endo H<sub>f</sub> untreated samples is labeled as gp160*. (b) The gp160 trafficking to TGN. The ratio of gp160r/total gp160 during the 2-hour chase period, normalized to NL4-3.</p>