Figure 6.

AUF1 and HuR associate with VEGFA mRNA in vivo and regulate VEGFA mRNA levels.(A) pVHL-positive RCC cells (786-O G7F) and pVHL-negative RCC cells (786-O 157Δ) were cultured either under normoxic conditions (room air) or hypoxic conditions (1% O₂) for 18 hours. Total cellular RNA was prepared and quantitative real time RT-PCR reactions were performed with VEGFA-specific PCR primers. VEGFA expression levels were determined in triplicate and averages are expressed relative to 18S rRNA levels. B, RNP-immunoprecipitations were performed from pVHL-negative 786-O 157Δ RCC cells with antibodies directed against AUF1, HuR, or pVHL, or an irrelevant rabbit IgG (rIgG). Immunoprecipitates were washed in lysis buffer, RNA was extracted, and RT-PCR was performed using VEGF-specific or actin-specific PCR primers. Products of PCR reactions were resolved on 2% low molecular weight agarose gels containing ethidium bromide and photographed under UV light. Reverse photographic images are shown. The VEGFA PCR primers amplify products of 567, 495, 363, and 267 base pairs due to expression of alternatively spliced forms of the VEGF mRNA, and the β-actin primers amplify a 600 base pair product (30).