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. 2002 Apr 17;3:3. doi: 10.1186/1471-2199-3-3

Figure 5.

Figure 5

Plx1 abolishes the Cdc14-binding activity of Net1N. Combinations of Plx1-phosphorylated or unphosphorylated Net1N and Cdc14 were tested for their abilities to form protein complexes. All four reactions consisted of identical components and permutations of four steps (1. addition of GST-Cdc14 beads; 2. incubation with Plx1 for 45 min at room temperature; 3. depletion of ATP at room temperature for 20 min; and 4. addition of Net1N). The ATP depletion step ensured that only the component(s) present with Plx1 prior to ATP depletion would be phosphorylated. Thus, Lanes 1 and 5 resulted from 1 -> 2 -> 3 -> 4; Lanes 2 and 6 resulted from 4 -> 2 -> 3 -> 1; Lanes 3 and 7 resulted from 1 -> 4 -> 2 -> 3; and Lanes 4 and 8 resulted from 1 -> 4 -> 3 -> 2. Whether Cdc14 or Net1 was phosphorylated by Plx1 in the presence of ATP (+) or unphosphorylated because of depletion of ATP (-), was indicated above each lane. All reactions were terminated by a final binding reaction at 4°C for 1 hr, and proteins in the supernatant (sup.) and bead fractions were separated by SDS-PAGE and immunoblotted with anti-T7 antibodies to detect both GST-T7-Cdc14 and His6-T7-Net1N.