FIG 4.

The maturation of the L protein is sequentially regulated by Hsp90 and the P protein. (A and B) At 24 hpi, Vero cells infected with MuV at an MOI of 5.0 were subjected to immunoprecipitation with anti-MuV V/P or anti-Hsp90 antibody and immunoblotting with the indicated antibodies. (C and D) 293T cells were transfected with pCAGGS-HA-MuV-L alone or cotransfected with pCAGGS-HA-MuV-L and pCAGGS-P, and the medium was replaced with fresh medium containing 0.5 μM 17-AAG at 12 h posttransfection. At 24 h posttransfection, the cell lysates were prepared using a urea-free cell lysis buffer and subjected to immunoblotting with the indicated antibodies. The relative band intensities of L protein were normalized to the β-actin level, and the relative expression levels of L protein based on the levels of DMSO-treated cells without P are shown (D). (E and F) At 24 h posttransfection, soluble and insoluble fractions of the cells were prepared as indicated in panel E and subjected to immunoblotting with the antibodies indicated in panel F. Error bars indicate the standard deviations of triplicates. The significance of differences between the means was determined by Student's t test. **, P < 0.01. ND, no significant difference.