Figure 3.

Deletion of the polypyrimidine tract in the HIF-1α 5′-UTR RNA strongly reduces its binding to PTB. (A) Schematic representation of the radiolabeled HIF-1α fragments. The polypyrimidine tract (PPT) is indicated as a black ellipse. (B) Cytoplasmic extracts from HEK293T cells overexpressing E-tagged c-Raf (−) or PTB (+) were incubated with radiolabeled HIF-1α IRES RNA or radiolabeled RNA corresponding to the HIF-1α IRES fragments HIF-delPPT, HIF-del1 and HIF-del2. After UV-irradiation, samples were treated with an RNase cocktail. One part of the sample was analyzed by SDS–PAGE (upper panel), and the other was mixed with anti-E-tag antibody for immunoprecipitation of labeled E-tagged PTB. Bound proteins were analyzed by SDS–PAGE. The arrow depicts the position of crosslinked E-tagged PTB.