Figure 4.

MU_NΔ/U_CΔH dimer formation in the course of reconstitution and the effect of DTT on its conversion to spliced product. (A) Dissociation of MU_NΔ/U_CΔH dimer formed during the reconstitution of MU_NΔ and U_CΔH to yield U_CΔH when treated with DTT in SDS prior to electrophoresis. MU_NΔ and U_CΔH were reconstituted in the absence of thiols, subjected to SDS/PAGE without (lane 2) or with DTT (lane 3) in the sample buffer, and determined by Western blot analysis using anti-His-tag antibody. (B) Dependence of protein splicing on DTT concentration. Mixtures of MU_NΔ and U_CΔH, reconstituted in the absence of DTT, were supplemented with DTT at the concentrations indicated and incubated at 25°C and pH 7.5 for 6 hr, followed by SDS/PAGE without additional thiol in the sample buffer and Western blot analysis with anti-His-tag antibody.