Depletion of E3α from rabbit reticulocyte extract by an anti E3α antiserum. Rabbit reticulocyte fraction II (50 μg) was mixed with buffer, preimmune IgG (3 μg), or anti-E3α IgG (3 μg), and rotated at 4°C for 30 hours. Protein A agarose (3 μL) was added to each sample and incubated an additional 3 hours at 4°C. After sedimentation of the agarose beads, the supernatant and washed beads were separately analyzed. (a) Anti-E3α immunoblot of postimmunoprecipitation supernatants. Supernatants of the above reactions were subjected to 8% SDS-PAGE, probed with anti E3α IgG at a dilution of 1:2000. Treatment of the extract with anti E3α IgG specifically removed a 200-kD immunoreactive band. (b) E3α activity was depleted from the postimmunoprecipitation supernatants after treatment with anti-E3α IgG. Supernatants of the above reactions were supplemented with AMPPNP (2 mM), Ub (25 μM), Ub aldehyde (1 μM), E1 (0.1 μM), E2_14k (5 μM), and ¹²⁵I-α-lactalbumin (∼0.75 μM). In the presence of LysAla (2 mM), a competitive inhibitor of E3α, no Ub-conjugation activity is seen (lanes 4–6). In the presence of the isomeric dipeptide, AlaLys (2 mM), which does not inhibit E3α, ¹²⁵I-α-lactalbumin-Ub conjugates were formed after buffer addition (lane 1) or after immunoprecipitation with preimmune IgG (lane 2). However, after immunoprecipitation with anti-E3α IgG, most of the E3α activity was abolished. (c) Pellets after anti-E3α IgG immunoprecipitation contained E3α activity. Washed protein A agarose beads from the above reactions (4-fold increase in amounts) were supplemented with AMPPNP (2 mM), Ub (25 μM), E1 (0.1 μM), E2_14k (10 μM), and ¹²⁵I-α-lactalbumin (∼0.75 μM). Only the beads that contained anti-E3α IgG supported formation of ¹²⁵I-α-lactalbumin-Ub conjugates.