FIG. 2.
HIV-1 RT expression and incorporation into VLPs. 293T cells were cotransfected with 10 μg pGAG and 10 μg of PR-defective GPfs, GPfsPR−, or the indicated construct. Cells and supernatant were collected at 48 h posttransfection for protein analysis as described in Materials and Methods. Cell samples corresponding to 4% of total cell lysates and supernatant samples corresponding to 50% of total recovered viral pellets were fractionated by 10% SDS-PAGE. (A) HIV-1 RT and Gag-Pol were probed with an anti-RT polyclonal antibody. Since R182 is smaller in size, we must consider the possibility that it was electrophoresed off the gel (lane 17). However, even when we repeated this experiment and carefully monitored the gel, we still failed to detect R182. (B) HA-tagged RT proteins were detected with an anti-HA monoclonal antibody. Membranes from top panels A and B were stripped and reprobed using a monoclonal antibody directed against HIV-1 p24CA. Note that the Pr160gag-pol bands (B, lanes 1 and 10) did not appear when probed with the anti-HA antibody but were detected by the anti-p24CA antibody. Molecular size marker positions are shown on the right.