Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: TOE1 is an inhibitor of HIV-1 replication with cell-penetrating capability

doi: 10.1073/pnas.1500857112

Figure Lengend Snippet: TOE1 peptides directly interact with HIV-1 TAR sequence. (A) RNA EMSA of a fluorescent TAR RNA probe in the presence of TOE1 peptides. Increasing amounts of either purified recombinant His-tagged 329–363 (Upper Left) or a synthetic peptide ED35 consisting of amino acids 329–363 of TOE1 (Upper Right) were incubated for 15 min at room temperature with TAR and then run on native 6% acrylamide gels. Specific competitor unlabeled RNA was added at the molar excess indicated. EMSA performed with a mutant TAR lacking the UCU bulge (Lower Left) and with WT TAR in the presence of mutant or WT unlabeled competitor (Lower Right) at 10× molar excess. (B) Electropherograms of labeled TAR with TOE1 synthetic peptides separated by capillary electrophoresis and detected by laser induced fluorescence after incubation at 75 °C for 5 min followed by a 5-min incubation at room temperature. The ER19 synthetic peptide consists of amino acids 329–347. (Left) TAR incubated with ER19 (0, 5, 7, and 9 µM). (Right) TAR incubated with ED35 (5, 7, and 9 µM). The complex between each peptide and TAR is observed as a peak at 6 min, and free TAR is observed at 7 min. All samples were also incubated with BSA and yeast tRNA and were separated under 500 V/cm in a 60-cm-long capillary in 25 mM Borax buffer at 22 °C.

Article Snippet: TAR/TOE1 complexes were visualized on a BioRad GelDoc+ apparatus with ImageLab software following separation on 6% acrylamide gels.

Techniques: Sequencing, Purification, Recombinant, Incubation, Mutagenesis, Labeling, Electrophoresis, Fluorescence