Fig. 3

ABIN1 depletion stimulates the activity of HIV-1 promoter (HeLa cells). a–e HeLa cells were transfected with siNC, siABIN1-1, or siABIN1-2 for 36 h, then cells were challenged with VSV-G pseudotyped luciferase reporter HIV-1(Luc). Cells were harvested at the indicated time points post infection for luciferase activity analysis, DNA or RNA extraction. a Luciferase activities of the infected cells were monitored at 48 hpi (upper panel) and ABIN1 knockdown efficiency was determined by Western Blots as the representative (lower panel). b–d Total DNA of the infected cells were extracted and quantified for subsequent real-time PCR at b 12 hpi for late RT levels, c 24 hpi 2-LTR-circle levels, d 48 hpi proviral DNA levels. e Total RNA was extracted at 48 hpi, and used for quantitation of HIV-1 mRNA. f The effect of overexpressed ABIN1 on LTR promoter activity were determined by luciferase assays after co-transfection of Flag-ABIN1 expressing plasmid or vector control together with pNL4-3.Luc.R-.E-, and pRL-TK in HEK-293T cells for 24 h. g Expression of Flag-ABIN1 was determined by Western Blots. The real-time PCR analysis of DNA and RNA extract were normalized to cellular β-globin and GAPDH, respectively. The primer pairs used were as described in “Methods”. Data are represented as mean ± SD of triplicate samples and are representative of at least three independent experiments. p values were calculated based on unpaired t test and significant changes relative to siNC or NC indicated. *p < 0.05; **p < 0.01; ***p < 0.001, ns not significant