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. 2011 May 2;286(26):22758–22768. doi: 10.1074/jbc.M111.235077

FIGURE 2.

FIGURE 2.

Depletion of CBC abrogates Tat transactivation of the HIV-1 LTR CAT reporter gene and provokes accumulation of nascent transcripts within the body of the gene. A, relative quantity of CBP20 mRNA was determined by RT-qPCR using total RNA isolated from HL3T1 cells that expressed F.Tat and were treated with the control (white bars) or CBP20 siRNA (black bars). Absolute mRNA levels were normalized to GAPDH mRNA levels. B, CAT assay of WCEs of HL3T1 cells containing an integrated HIV-1 LTR CAT reporter gene is shown. Cells were treated with the control (white bars) or CBP20 siRNA (black bars) and expressed F.Tat as indicated below the CAT data. C, relative quantity of CAT mRNA was determined by RT-qPCR using total RNA samples isolated from HL3T1 cells that expressed F.Tat and were treated with the control (white bars) or CBP20 siRNA (black bars). Absolute CAT mRNA levels were normalized to the levels of transfected F.Tat gene. D, qNARIP was used to analyze HIV-1 LTR CAT nascent transcripts in HL3T1 cells expressing F.Tat. Cells were treated with the control (white bars) or CBP20 siRNA (black bars). Nascent mRNA was immunoprecipitated with the RNAPII antibody. Results are presented as percent of input DNA. Two different primer pairs were used for the amplification of transcripts at the 5′ region (5′r) or downstream region (Dr) as indicated below the schematic of the HIV-1 LTR CAT reporter gene. Arrow and pA depict transcription start site and polyadenylation signal, respectively. Primer pairs used for the amplification of DNA in ChIP-qPCR analysis (Fig. 3) at the promoter region (Pr) or coding region (Cr) are indicated above the gene schematic. Numbers below the schematic represent nucleotide positions of the gene in respect to the transcription start site. E, qNARIP was used to analyze HIV-1 nascent transcripts in HeLa expressing F.Tat and HIV-1 genome from pNL4–3 plasmid. Cells were treated with the control (white bars) or CBP20 siRNA (black bars). Nascent mRNA was immunoprecipitated with the RNAPII antibody. Results are presented as percent of input DNA. Four different primer pairs were used for the amplification of transcripts at the 5′ region (5′r) or downstream regions (Dr1, Dr2, and Dr3) as indicated below the schematic of the HIV-1 genome. Arrow and pA depict transcription start site and polyadenylation signal, respectively. Numbers below the schematic represent nucleotide positions of the gene in respect to the transcription start site.