Figure 4.

SCIRT interacts with HuR to stabilize the VEGFA mRNA through ubiquitin-proteasome pathway under HR condition. (a) RNA FISH revealed that SCIRT is predominantly localized in the nuclear of HUVECs, while being exported to the cytoplasm with HR treatment (scale bar, 50 μm). (b) Identification of proteins that associate with SCIRT. Biotinylated SCIRT or biotinylated antisense SCIRT was incubated with HUVEC extracts; RNA pull-down combined was performed to identify the proteins that associate with SCIRT. (c) HuR was identified specifically bind to SCIRT by western blot. (d) RIP was performed to confirm the interaction of HuR and SCIRT. (e) The protein level of HuR determined by western blot was positively regulated by SCIRT, however, which was not affected by variation of VEGFA. (f) Overexpression and knocking down of SCIRT have no effect on RNA levels of HuR; HuR was regulated by SCIRT post-translationally. (g) HUVECs, transfected with the siRNA of SCIRT, were treated with CHX (125 μg/ml), an inhibitor of protein synthesis. The protein levels of HuR and VEGFA were detected by western blot after 1, 2, and 3 h. (h) Western blot analysis of HuR in the SCIRT knockdown cells treated with DMSO, MG132, or RAP under HR conditions. (i) HUVECs transfected with SCIRT siRNA were treated with MG132 (5 μM) for 24 h. Cell lysates were immunoprecipitated with either control IgG or an antibody against HuR and analyzed by immunoblotting with a ubiquitin- (Ub-) specific antibody. Bottom, input from cell lysates.