HCV protein and RNA detection in immortalized mouse hepatocytes. (A) Western Blot detection of HCV NS5A. Cell lysate was harvested from individual cell clones and resolved by SDS-PAGE. Samples include cell lysate from Huh-7 sgCon1 (lane 1); non-transfeted (NT) Huh-7 (lane 2); Huh-7 sgJFH-#2 (lane 3); NT MMHD3 cells (lane 4); MMHD3 sgJFH#1 (lane 5); and MMH1-1sgJFH#4, 5, 6, 7, 9, and 10 (lanes 6–11). (B) Northern Blot detection of sgJFH-1 RNA. Total RNA was isolated from non-transfected (NT) and individual MMHD3 (lanes 2), MMH1-1 (lanes 4–9), and Huh-7 (lanes 11–15) sgJFH-1 replicon clones. Positive-strand (top panel) and negative-strand (middle panel) HCV RNA was detected with 32P-labeled strand-specific riboprobes. Cellular GAPDH was detected with a 32P-labeled cDNA probe (bottom panel). Serial dilutions of in vitro transcribed positive-strand and negative-strand sgJFH-1 RNA (109, 108, 107) are shown in lanes 16–18 and lanes 19–21, respectively.