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Figure 2 | Retrovirology

Figure 2

From: NUCKS1, a novel Tat coactivator, plays a crucial role in HIV-1 replication by increasing Tat-mediated viral transcription on the HIV-1 LTR promoter

Figure 2

NUCKS1-mediated Tat activation on HIV-1 LTR. (A) One hundred nanograms of the Flag-Tat and/or 3 μg of the NUCKS1 expression plasmid was transfected with 200 ng of the HIV-1 LTR-driven luciferase reporter (pGL3-LTR-Luc) and 20 ng of pCMV-LacZ as a transfection control into HeLa cells. At 24 h after transfection, luciferase activity was measured and normalized to β-galactosidase activity. The data are expressed as mean ± SD (n = 3). *, P < 0.01 as compared with the Flag-Tat transfected only. Expression levels of NUCKS1 and Tat were assessed with Western blotting. (B) Luciferase activity was assessed in HeLa cells 24 h after cotransfection of pGL3-LTR-Luc (200 ng), Flag-Tat (100 ng), and increasing amounts of V5-NUCKS1 (0, 0.5, 1, 2, and 3 μg), together with 20 ng of pCMV-LacZ as a transfection control. The data are expressed as mean ± SD (n = 3). *, P < 0.01 and **, P < 0.05, as compared with the Flag-Tat transfected only (C) Knockdown of NUCKS1 was performed with three siRNAs against NUCKS1 mRNA. HeLa cells were transfected with control siRNA or three NUCKS1 siRNAs. At 48 h after transfection, knockdown of NUCKS1 by siRNA was assessed by Western blotting using anti-NUCKS1, anti-Flag and anti-β-actin antibodies as a loading control. (D) NUCKS1-engaed Tat activity was assessed by the luciferase assay combined with the knockdown experiment. HeLa cells were transfected with control siRNA or three NUCKS1 siRNAs. Twenty-four hours after transfection, cells were transfected further with pGL3–LTR–Luc, pCMV-LacZ, and Flag-Tat. The luciferase assay was conducted as described in A. The data was expressed as mean ± SD (n = 3). *, P < 0.01 as compared with the cells transfected with control siRNA.

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