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. 2010 Sep 7;78(11):4532–4541. doi: 10.1128/IAI.00802-10

FIG. 2.

FIG. 2.

Restoration of ARAP2 expression allows normal entry of Listeria in the presence of ARAP2 siRNA. (A) Expression of an ARAP2 cDNA that is resistant to siRNA-mediated depletion. HeLa cells were transfected with either control (C) siRNA or ARAP2-1 (A2-1) siRNA, which targets the 3′ untranslated region (UTR) present in endogenous ARAP2 mRNA. Cells were transfected again with plasmids expressing either Flag-tagged luciferase (L) or ARAP2 (AR2) encoded by a cDNA that lacks the 3′ UTR. At about 24 h after plasmid DNA transfection, ARAP2 expression was evaluated by Western blotting. Note that ectopic expression of ARAP2 lacking the 3′ UTR (AR2) restored normal ARAP2 levels to cells treated with ARAP2-1 (A2-1) siRNA. (B) Levels of Flag-tagged luciferase or ARAP2 expression. Confocal microscopy was used to measure relative expression of tagged luciferase or ARAP2 proteins in transfected HeLa cells. P = 0.19 (not significant). (C) Restoration of bacterial entry in cells expressing ARAP2 resistant to siRNA-mediated depletion. A fluorescence microscopy-based assay (38) was used to measure entry of Listeria in cells transfected with siRNAs and expressing Flag-tagged luciferase or ARAP2. Values are averages ± SD from three experiments. *, P < 0.001 compared to the luciferase (L) plus control siRNA (C) condition.