FIGURE 4.

Alpha4ΔC binds PP2A but fails to bind Mid1. A, HEK293FT whole cell lysate was incubated with cobalt-nitrilotriacetic acid resin in the absence (−) or presence (+) of either Alpha4ΔC or Alpha4ΔC_AA (mutation of the PP2Ac-binding residues). Bound proteins were eluted with 200 mm imidazole, and the eluate was analyzed by SDS-PAGE and immunoblotting with antibodies recognizing PP2Ac (upper panel) and the His₆ tag (lower panel). Data are representative of three independent experiments. WB, Western blot. B, HEK293FT cells were transfected with HA₃-PP2Ac, FLAG-Alpha4, FLAG-Alpha4ΔC, Myc-Mid1, or a combination of the constructs. FLAG immune complexes (Flag IPs) were isolated from the cells and subjected to SDS-PAGE and Western analysis using anti-Myc, anti-PP2Ac, and anti-Alpha4 antibodies.