Fig. 4

Presence of HIV-1 RNA associated complexes in multiple HIV-1 infected whole cell extracts. a Fresh primary PBMCs (10⁷ cells) were cultured with PHA/IL-2 for 7 days and infected with HIV-1 89.6 strain (MOI:1) [7]. Three days later they were treated with F07#13 (once every other day at 0.1 µM) for a total of 20 days. Cells were collected and lysates were loaded onto a sizing column under high salt. Column fractions were then IPed with antibodies against PRC2, Sin3A, Cul4B, and IgG. Following IP, RNA was isolated and samples were processed for RT-qPCR using primers against TAR-gag. Non-specific IgG background IPs were used as a control. Fractions from Complexes I, II, and III (500 µl) of infected PBMCs were nanotrapped with NT084 and assayed for RT-qPCR for presence of 7SK RNA (b) or half of the samples were run on an SDS/PAGE and Western blotted for presence of PRC2, Cul4B, actin, and Sin3A (data not shown) (c). Fraction 10 was used as a control in lane 1 of panels b and c. Error bars represent ± SD of three technical replicates