FIG. 7.
RC incorporation of NS5B from a cyclosporine-resistant replicon was resistant to cyclosporine. (A) GS5 and RS1-2 replicon cells were treated in parallel with cyclosporine (CsA) and PK (0.25 μg/ml) as indicated. The cell lysates were then subjected to the detection of NS5B and Grp94. The anti-Grp94 antibody also recognized an additional band of higher-than-expected molecular weight that was completely removed by PK treatment (not shown). Proteins isolated from 4 × 105 cells were loaded into each lane. (B) Dosage-dependent effect of cyclosporine on the PK sensitivity of the GS5, but not the RS1-2, NS5B protein. Experiments described above (A) were performed with 0 to 8 μg/ml of cyclosporine. The protein bands detected by Western blotting were quantified, normalized to Grp94 for loading and PK digestion, and then plotted as the ratio of the protein level in the cyclosporine-treated sample to that in the untreated sample. (C) The CRC incorporation of NS5B in RS1-2 cells was resistant to cyclosporine. Isolation of the MF and digestion of the CRC were performed with RS1-2 cell lysates as described in the legend of Fig. 3. The loading amounts of the different fractions were obtained from 1 × 106 cells for total lysate and the MF and from 2.5 × 106 cells for CRC-PK.