Fig. 4

ABIN1 suppresses HIV-1 Tat ubiquitination via its ubiquitin binding property. a HEK-293T cells were transfected with siRNAs targeting ABIN1 (siABIN1-1, siABIN1-2) or siNC for 24 h, then a second round transfection of Flag-Tat expressing constructs was performed for another 24 h, and cells were then harvested and assayed as described in “Methods”. b After transfection with Myc-ABIN1 and Flag-Tat expressing plasmids for 24 h  as described in “Methods”, the ubiquitination of Tat in HEK-293T cells was analyzed by Flag IP under denaturing conditions as in (a). c HEK-293T cells were co-transfected with plasmids encoding Myc tagged wild-type ABIN1 or ABIN1-QE2 mutant and Flag-Tat for 24 h, cells were then harvested and analyzed as in (a). d The effect of overexpressed ABIN1 and QE2 mutant were determined by luciferase assays after HIV-1(Luc) infection following ABIN1, QE2 or vector control (NC) transfection in HeLa cells. The panel below indicates the expression of ABIN1 or its mutant. β-actin was detected as sample loading control. Data are represented as mean ± SD of triplicate samples, all data and Western Blots are representative of at least three independent experiments. p values were calculated based on unpaired t test and significant changes relative to NC indicated. *p < 0.05; **p < 0.01; ***p < 0.001