Figure 10. Purification of recombinant MPDE3B expressed in Sf21 cells: interactions between purified recombinant MPDE3B and recombinant PKB, pPKB and p-ΔPKB during gel filtration on Superose 12.

(A) Left panel: Sf21 cell lysates infected without (lane 2) or with (lane 3) MPDE3B-baculovirus; (lane 4) FLAG-tagged MPDE3B purified from Sf21 cell lysates as described in the Materials and methods section by anti-FLAG affinity chromatography (specific activity 0.71±0.16 μmol·min⁻¹·mg⁻¹, n=4) or (lane 5) (from another experiment) recombinant pPKB (Upstate Biotechnology) were subjected to SDS/PAGE. Gels were stained with Simply Blue Safe stain. Right panel: lanes 6–8: proteins from gels similar to that on the left (lanes 2–4) were electrophoretically transferred to NC membranes and immunoblotted with anti-FLAG peroxidase-conjugated antibodies for immunodetection of purified MPDE3B (lanes 7 and 8). (B, C) Purified recombinant WT PDE3B (0.25 or 1 μg, ∼2 or ∼8 pmol, based on predicted molecular mass of ∼122000) and PKB, pPKB or p-ΔPKB (500 ng, ∼9 pmol, based on predicted molecular mass of ∼55000) were incubated (30 min, 30 °C) in buffer C (total volume, 200 μl) with BSA (50 μg/ml), before dilution by addition of 800 μl of buffer C with BSA (50 μg/ml). Samples (1 ml) were then applied to Superose 12 HR 10/30 equilibrated and were eluted with buffer C and BSA (50 μg/ml). Fractions (0.5 ml) were collected on Frac 950 and portions were assayed for PDE3 and PKB activities (results not shown). Protein contents were monitored at A₂₈₀ by AKTA monitor UPC 900. Portions (20 μl) of indicated fractions (0.5 ml) were subjected to SDS/PAGE, and immunoblotting with antibodies specific to PKB, pPKB and PDE3B-CT (B) and p-ΔPKB and PDE3B-CT (C). The results are representative of two experiments.