Figure 3
FIP3 is phosphorylated on S102 in a cell cycle-dependent manner. Panel A 200 ng of His6-tagged recombinant FIP3, or 200 ng His6-tagged recombinant FIP3 incubated with Cdk1-cyclin B to phosphorylate the protein on S102 (see below) were separated by SDS-PAGE, transferred to nitrocellulose and analysed by immunoblotting using 0.2 μg/ml affinity-purified anti-pS102 antibodies, incubated in the presence of either the phosphorylated or non-phosphorylated peptide (2.5 μM) as indicated. The position of molecular weight markers (kDa) is shown. Data from a typical experiment are presented, repeated four times with similar results. Panel B HeLa cells expressing GFP-FIP3 (or control cells) were lysed as described in Methods. Samples of lysate were separated by SDS-PAGE, transferred to nitrocellulose and probed with anti-pS102 antibodies. GFP-FIP3 is readily detected (endogenous FIP3 is expressed at too low levels to be readily detected by this antibody). Panel C HeLa cells stably expressing GFP-FIP3 were synchronised using a thymidine and nocodazole block as described in Methods. Cells were harvested 30, 60, 90 or 120 min after release from the block, corresponding to metaphase (~30 min) through late telophase (120 min)[11], fractionated into membrane and cytosolic fractions, and these fractions together with the initial homogenate (total) were immunoblotted with anti-GFP (to reveal total GFP-FIP3 levels in each fraction), anti-pS102 and anti-cyclin B as shown. Data from a typical experiment is presented, repeated three times with quantitatively similar results, which are quantified in Panel D (the signals at each point and in each fraction are expressed as a% of that in the 30 minute total cell lysate level. The mean of triplicate experiments (± S.D.) is shown).