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Fig. 3 | Retrovirology

Fig. 3

From: Effect of transcription inhibition and generation of suppressive viral non-coding RNAs

Fig. 3

TAR-gag RNA association with various inhibitory complexes. a Early–mid log phase HIV-1 infected J1.1 cells were treated with F07#13 for 48 h (1 μM), pelleted, washed (×2) with PBS without Ca2+ and Mg2+, resuspended in lysis buffer, and 2500 µg of protein were equilibrated in degassed FPLC running buffer. A Superose 6 10/300 size-exclusion chromatography column was used to run lysed samples. Fractions were then pre-cleared with IgG for 2 h at 4 °C and then divided into 4 sub-fractions for IP using six antibodies against PSMD11, Sin3A, PRC2, HDAC-1, DNMT3A, and Cul4B (5 μg/reaction). Protein A/G was added the next day and the IPed complexes were washed. RNA was isolated for RT-qPCR using TAR-gag primers. An IP with IgG antibody was used as a control. Fractions from Complexes I, II, III, and IV constitute complex sizes from ~ 2.2 MDa to ~ 300 kDa. Error bars represent ± SD of three technical replicates. b Fractions from Complexes I, II, and III (500 µl) were nanotrapped with NT084 and assayed for RT-qPCR for presence of 7SK RNA. Fraction 10 was used as a control in lane 1 of this panel

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