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. 1998 Jul;72(7):5638–5647. doi: 10.1128/jvi.72.7.5638-5647.1998

FIG. 9.

FIG. 9

IRNA binds proteins that interact with HCV 5′ UTR. (A) 32P-labeled IRNA (lanes 1 to 3) and HCV 5′ UTR RNA (lanes 4 and 5) were UV cross-linked to cellular polypeptides, using 30 μg of HeLa S10 fraction (lanes 2 and 4) and 0.3 μg of purified La protein (lanes 3 and 5). Numbers to the left correspond to the molecular masses (in kilodaltons) of the polypeptides indicated. Lane 1 contains the IRNA probe but no S10 extract. (B) Competition UV-cross-linking studies were performed with 32P-labeled HCV 5′ UTR RNA and 15 μg of HeLa S10 in the absence (lane 2) and presence of various unlabeled competitor RNAs (lanes 3 to 7). Lanes 3 and 4, 250- and 500-fold molar excess of unlabeled HCV 5′ UTR; lanes 5 and 6, 250- and 500-fold molar excess of unlabeled IRNA; lane 7, 500-fold molar excess of a nonspecific RNA (polylinker region of pSPluc; Promega). Lane 1 contains the probe but no S10 extract; lane M shows the migration (in kilodaltons) of marker proteins. The numbers to the right correspond to the molecular masses (in kilodaltons) of proteins which cross-link to the labeled HCV 5′ UTR probe. (C) 32P-labeled IRNA (lanes 1 to 4) and HCV 5′ UTR RNA (lanes 5 to 8) were UV cross-linked to cellular polypeptides, using 30 μg of S10 extract of either HeLa cells (lanes 2 and 6), Huh7 cells (lanes 3 and 7), or Huh7 cell line pCDIR.Ribo.ΔT7 (lanes 4 and 8). Lanes 1 and 5 contain the probe but no S10 extract. Numbers to the right correspond to the molecular masses (in kilodaltons) of the polypeptides indicated. Lane M shows the migration (in kilodaltons) of marker proteins. (D) Competition UV-cross-linking studies were performed with 32P-labeled HCV 5′ UTR RNA and purified La protein (150 ng) in the absence (lane 1) and presence of 100-fold (lanes 2, 4, and 6) and 200-fold (lanes 3, 5, and 7) molar excesses of unlabeled IRNA (lanes 2 and 3), nonspecific RNA (lanes 4 and 5), and HCV 5′ UTR RNA (lane 6 and 7). Lane M shows the migration (in kilodaltons) of marker proteins. The migration of La protein cross-linked with HCV 5′ UTR is indicated.