(A) Expression of Rdh12 and its mutants. Lanes were loaded with extracts from Sf9 cells infected with virus only, virus containing RDH12, T49M, R161Q, A177V, C201R, and A269fsX270 from left to right, respectively. Immunoblotting was carried out according to standard protocols using Immobilon-P to adsorb proteins (polyvinylidene difluoride; Millipore Corp.) (Ohguro et al., 1993). Mouse polyclonal antibodies against bacterially expressed full-length mouse Rdh12 were raised in BALB/c mice as described previously (Maeda et al., 2006). Secondary antibody is HRP conjugated anti-mouse IgG (Promega Corporation, Madison, WI) and signal was detected using SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology, Rockford, IL). (B) Calculated Km and Vmax values of Rdh12 constructs expressed in Sf9 cells. ND, not determined.