Fig. 2. Conjugation of DmSmt3 to DmUba2 and DmUbc9 in vitro. Fly embryo extracts supplemented with ATP and Mg²⁺ were incubated with His₆DmSmt3, His₆DmUb, or no tagged protein, and proteins were isolated using Ni-NTA beads and analyzed by SDS-PAGE and immunoblotting (see Materials and Methods). (A,B) Immunoblotting using DmUba2-specific antibodies. SDS-PAGE was conducted under reducing (A) or nonreducing (B) conditions. In (A), a sample of unpurified lysate was also analyzed. The species migrating at 55 kDa is presumably a breakdown product of DmUba2 (see also Fig. 1A,B). (C,D) Immunoblotting using DmUbc9-specific antibodies. SDS-PAGE was conducted under reducing (C) or nonreducing (D) conditions. In C, a sample of unpurified lysate was also analyzed. In addition to DmUbc9 (predicted molecular weight, 18 kDa) (Joanisse et al., 1998), anti-DmUbc9 antibodies also recognize a polypeptide of 25 kDa; as this species is evident both in the lysate and among proteins isolated with His₆DmUb, it may be a DmUb-conjugating enzyme that is related to DmUbc9. Molecular weight markers are shown for each panel.