FIGURE 5.

The E2s may act as dimers. A, spontaneous dimerization profile of E2–25K (left), E2T (middle), and E2C (right) captured by cross-linking with DTSSP. Reduced and non-reduced equal portions of the cross-linked mixtures were separated on SDS-PAGE and analyzed by Western blot with anti-His. The entire repertoire of the E2 dimerization profile is presented in supplemental Fig. S10. B, both the monomeric and dimeric forms of the E2 can acquire ubiquitin. Following cross-linking, E2-25K, E2T, and E2C were separated to dimeric and monomeric forms on non-reducing SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was incubated with E1 and wild type ubiquitin in standard ubiquitination conditions. Charging of the monomeric and dimeric forms was determined by Western blot analysis with anti-ubiquitin. IB, immunoblot.