FIGURE 5.

Both the Mid1-binding domain and PP2Ac binding are essential for Alpha4 inhibition of PP2Ac polyubiquitination. HEK293FT cells were transfected with HA₃-PP2Ac and empty vector, full-length WT FLAG-Alpha4, FLAG-Alpha4ΔC, or FLAG-Alpha4_ED. At 48 h post-transfection, cells were treated with 25 μm proteasome inhibitor (MG132) for 4 h at 37 °C prior to lysis. Total polyubiquitinated proteins were isolated from the cell lysates using TUBE2-agarose beads. Protein expression and the polyubiquitination state of ectopic PP2Ac were analyzed via immunoblotting using anti-PP2Ac, anti-Alpha4, and anti-ubiquitin antibodies.