Figure 2.

Differential localisation of the HuR protein between TECs and NECs. HuR localisation was analysed by fluorescent immunocytochemistry. HuR was localised only in the nucleus of HMVEC, whereas it was expressed in the cytoplasm as well of oral carcinoma and melanoma cells (A). HuR expression was limited to the nuclei of mouse normal ECs (skin EC); however, it was detected not only in the nucleus but also in the cytoplasm of mouse tumour ECs (oral carcinoma and melanoma ECs) (B). Blue: DAPI, Green: HuR (white bar: 50 μm). To confirm the cytoplasmic localisation of HuR in TEC, ECs were separated into cytoplasmic and nuclear fractions. Using two antibodies, anti-hnRNP (nuclear protein) and anti-β-tubulin (cytoplasmic protein), it was confirmed that nuclear and cytoplasmic protein extracts were pure in each fraction. The HuR of each fraction was detected by western blotting. The amounts of HuR in the cytoplasm of TECs were much higher than NECs (^*P<0.05 vs skin EC) (C and D). HuR expression was analysed in human tissue sections. HuR was expressed only in nuclei in human NECs (white arrow); however, it was expressed in cytoplasm in human TECs (yellow arrow), which were stained by anti-CD31 antibody (E). (Black bar: 50 μm) Immunofluorescent double staining with anti-CD31 and anti-HuR antibodies in the frozen sections of human renal tumours and normal renal tissues. HuR staining is stained in cytoplasm in TECs stained with anti-CD31, but only in nuclei in NEC (F). (White bar: 10 μl).