Figure 2. Localization of human antigen R after gene silencing. The normal corneal fibroblasts were treated with GAPDH siRNA, β-actin siRNA, or human antigen R (HuR) siRNA. The immunoreactivity of HuR was analyzed at 72 h post-transfection. A: Fibroblasts stained with Hoechst nuclear stain after a treatment with GAPDH siRNA. B: Immunoreactivity of fibroblasts with anti-HuR antibody after a treatment with GAPDH siRNA. C: Overlay of (A) and (B). D: Fibroblasts stained with Hoechst nuclear stain after a treatment with β-actin siRNA. E: Immunoreactivity of fibroblasts with anti-HuR antibody after a treatment with β-actin siRNA. F: Overlay of (D) and (E). G: Fibroblasts stained with Hoechst nuclear stain after treatment with HuR siRNA. B: Immunoreactivity of fibroblasts with anti-HuR antibody after treatment with HuR siRNA. C: Overlay of (G) and (H). Note that the GAPDH siRNA and β-actin siRNA treatments had no effect on HuR expression and its nuclear localization. HuR gene silencing significantly reduced the expression of HuR, as shown by the absence of green fluorescence in nuclei.