Cyclosporine Inhibits a Direct Interaction between Cyclophilins and Hepatitis C NS5A
Figure 6
Mutant NS5A binds CypB better than wild-type NS5A in cells.
(A) 293T cells were co-transfected with either wt-NS5A-His (lanes 2 and 3) or mt- NS5A-His (lanes 4 and 5) together with HA-tagged CypB in the presence and absence of 2.0 µg/ml CsA. Forty eight hours after transfections, cell lysates were immunoprecipitated with anti-His antibody. The immunoprecipitates (IP) were Western blotted and probed with NS5A-specific and HA-specific antibodies to detect NS5A and CypB respectively. Lane 1 represents transfections with an empty vector control. (B) 293T cells were co-transfected with either HA-wt NS5A (lanes 3 and 5) or HA-mt NS5A (lanes 2 and 4) in the presence (lanes 4 and 5) or absence (lanes 1–3) of FLAG-wt NS5B (Con 1b). Lane 1 represents transfections with an empty vector control. Forty eight hours after transfections, cell lysates were immunoprecipitated with anti-HA antibody and Western blotted. The blots were probed with antibodies specific for NS5A (top blot), FLAG (to detect NS5B (middle blot), and CypB (lower blot) respectively. Upper two blots show total cell lysate without HA-specific immunoprecipitation.