CTGC motifs flanking the HIV TATA box are required for correct PICH formation in vitro and Tat trans -activation in living cells. EMSAs were performed as in Figure 2. (A) EMSA mobility comparison of complexes formed on HIVwt promoter (lane 1) and CTGC5’3’ mutant (lane 2). PICHs bound to HIVwt are indicated at the left of the panel. Complexes formed on CTGC3’5’ mutant are indicated at the right as aberrant pre-initiation complexes (aPIC) (B) For supershift assay with the CTGC5’3’ radiolabelled probe, no antibody (lanes 1 and 9) or the indicated antibodies (lanes 2–8 and 10–11) were added in the reaction. Arrowheads indicate supershifted bands. (C) Basal and Tat transactivated promoter activity and (D) Tat transactivation are measured by luciferase activity in extracts of HeLa cells as in Figure 6C and D. Mutations in the CTGC motifs are shown in shades of blue.