HEXIM1-Tat chimera inhibits HIV-1 replication (original) (raw)
Fig 2
HT1 prevents Tat from bringing active P-TEFb to TAR.
A. HT1 binds to P-TEFb and competes with Tat for P-TEFb binding. m:HT1 and/or f:Tat were transiently expressed in 293T cells. Cell lysates were used for immunoprecipitation (IP) using anti-Myc Ab (upper panel, lanes 2–7), anti-Flag Ab (middle panel, lanes 2–7), or control IgG (upper and middle panels, lane 1). Input lysates (lower panel) and immunoprecipitates were submitted to SDS-PAGE and WB using anti-CycT1, anti-Myc, anti-Flag and anti-actin Abs. B. HT1 inhibits the kinase activity of P-TEFb subunit CDK9. m:HT1 and/or f:Tat were transiently expressed in 293T cells. Cell lysates were used for IP using anti-Myc Ab (lanes 2–4), or control IgG (lane 1). Immunoprecipitates were incubated with ATP and recombinant GST-CTD proteins for in vitro kinase assay. Total GST-CTD was detected using anti-GST Ab and phosphorylated GST-CTD (CTD-P) was detected using anti-Ser2P Ab. m:HT1 and m:Tat were detected using anti-Myc Ab. Six replicate experiments were performed, and mean relative kinase activities are shown in the bar graph to the right. C. HT1 binds to TAR. m:HT1 or m:Tat (or empty vector, EV, as a control) was transiently co-expressed with TAR RNA-expressing pU16TAR in 293T cells. Cell lysates were used for IP using anti-Myc Ab or control IgG. RNA was purified from the immunoprecipitates and submitted to RT-qPCR using TAR-specific primers (upper left panel). Relative TAR enrichment was calculated as qPCR count using anti-Myc Ab minus using IgG, and normalized to EV. Error bars represent standard deviation from triplicate qPCR assays. Input lysates were submitted to SDS-PAGE and WB using anti-Myc and anti-Actin Abs (lower left panel). The amounts and standard deviations of immunoprecipitated TAR RNA (from the upper left panel) were normalized to the respective amount of m:HT1 or m:Tat detected in the input lysate (right panel). D. HT1 competes with Tat for TAR binding. m:HT1 and/or f:Tat were transiently co-expressed with TAR RNA-expressing pU16TAR in 293T cells. Cell lysates were used for IP using anti-Myc Ab or control IgG. RNA was purified from the immunoprecipitates and submitted to RT-qPCR using TAR-specific primers (upper panel). Relative TAR enrichment was calculated as qPCR count using anti-Myc Ab minus using IgG, and normalized to EV. Error bars represent standard deviation from triplicate qPCR assays. Input lysates were submitted to SDS-PAGE and WB using anti-Myc, anti-Flag and anti-Actin Abs (lower panel).