Design and validation of the TrwC/Rep chimeric protein.

(A) Schematic representation of Rep68, TrwC and the TrwC/Rep chimera. For each protein, overall configuration and functional domains are shown. The position of the HUH and Y2 motif, and the NLS (black bar) are indicated. Amino acid (aa) positions are indicated above each protein. (B) Western blot analysis of Rep68 and TrwC/Rep chimera expressed in 293T cells. Molecular weight in kDa is shown on the right. Antibodies used to detect proteins are shown on the left. (C) Localization of TrwC, Rep68 and TrwC/Rep chimera in 293T cells visualized by immunofluorescence microscopy. Images are shown at 60X magnification. (D) Assessment of the purity of His-Rep68 and His-TrwC/Rep chimera purified by nickel affinity and gel filtration chromatography. 200 ng of protein was loaded onto a 12% SDS-PAGE gel and subsequently stained with Coomassie Brilliant blue to visualize the quality of the purified protein. Marker (M) is shown on the left; molecular weight from top to bottom is: 250, 150, 100, 75, 50 kDa.