Figure 2.

The Ring finger domain of hMEX‐3C mediates specific E2‐dependent ubiquitination. (A) Purified Ring finger domain of hMEX‐3C was incubated with E1, ubiquitin and various E2 (UbcH5b, UbcH5c, and UBE2H) (Lanes 2, 3, and 4) or without E2 enzyme (Lane 1). All reaction mixtures were kept at 37°C for 90 min. Then, reaction mixtures were fractionated by 10% SDS‐PAGE and analyzed by immunoblotting using anti‐Ub and anti‐GST antibodies. (B) Each component is required for ubiquitination. In every lane, one component was removed in each reaction. Then the reaction mixtures were resolved by 10% SDS‐PAGE and analyzed by western blot with antibodies.