HIV-1 proteins exhibit altered trafficking to the plasma membrane in Ubc9 knockdown cells.
2013-07-08T02:06:47Z (GMT) by
<p>(a) 293T cells were transfected with pNL4-in combination with either Ctr. siRNA or Ubc9 siRNA. Cells were pulse labeled with [<sup>35</sup>S] methionine/cysteine for 30 minutes and chased for 1.5 and 2.5 hours. At indicated times, cells were ruptured and treated with Benzonase (50 units) for 0.5 hours. Lysates were centrifuged at 4°C, for 20 hours at 34,500 RPMs in a ML-130 rotor. Supernatants were carefully collected leaving behind the pellet containing cellular organelles and adjusted to 1X lysis buffer. Viral proteins were immunoprecipitated with patient serum, separated by SDS PAGE, and visualized by phosphorimaging using The Discovery Series Quantity One software. (b) Quantitation of viral proteins associated with the plasma membrane. gp120/Pr55 ratios of the PM fraction during the 1.5 hour and 2.5 hour chase times. (c) Steady state Env surface expression. Transfected cells were resuspended in PBS, fixed, and surface Env was assayed by flow cytometry using HIV-IG and Alexa Fluor-488 goat-anti-Human conjugated Antibodies.</p>