Analysis of PICs assembled on the HIV-1 promoter in vitro. A. Experimental strategy for analyzing active PICs. Step 1: Cell-free transcription reactions are performed using HeLa nuclear Extract (NE) and biotinylated templates carrying the HIV-1 LTR. Step 2: Transcription complexes are purified by binding to streptavidin-coated magnetic beads. Step 3: Nascent RNA chains are labeled by incorporation of α-[32P]UTP. A schematic representation of the HIV-1 double G-less cassette template used in the experiment is shown at the top of the panel. B. In vitro transcription reactions were performed with the purified PICs. Lanes 1-3 represent three independent reactions performed using the HIV-1 template. Arrows indicate the migration of long and short transcripts. C. TCERG1 is a component of the HIV-1 PICs. PICs were formed on constructs containing (lane 3) or not (lane 2) the HIV-1 promoter. PICs were purified with streptavidin-coated magnetic beads, and Western blot analysis of the purified PICs was performed using the specific antibodies shown at the right side of the panel. The relative mobilities (in kDa) of the molecular mass markers (M) are shown on the left side of the panel.