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Figure 2 | Retrovirology

Figure 2

From: The RNA binding protein HuR does not interact directly with HIV-1 reverse transcriptase and does not affect reverse transcription in vitro

Figure 2

Purification and biophysical characterization of GST- and NusA-HuR. (A) Analysis of the expression and purification of GST-HuR by SDS PAGE. Lanes 1 and 2 illustrate total protein and total soluble protein after E. coli cell lysis, respectively, lane 3 shows the flow-through fraction of the glutathione sepharose column, lane 4 contains molecular weight markers and lane 5 is the eluate during the column wash. Purified GST-HuR eluted from the glutathione sepharose column with 20 mM glutathione is shown in lane 6 and lanes 7-10 contain purified GST-HuR that was incubated with 0.15, 0.1, 0.05 and 0.03 units of thrombin, respectively, for 1 h at room temperature. Lane 11 contains purified GST-HuR incubated for 1 h in buffer at room temperature in the absence of thrombin. (B) Size-exclusion multi-angle light scattering analysis of NusA-HuR (black) and NusA-RRM 3 (grey). The elution profiles (circles) and the predicted molecular masses obtained from the light-scattering measurements (triangles) are shown. Both proteins were found to exist > 90% as monomers in solution. (C) Gel-shift assay of HuR binding to single-stranded RNA. The sequence of the RNA used in this experiment is shown above the autoradiograph. The ARE sequence is highlighted and underlined. HIV-1 RT and NusA were included as controls in this experiment.

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