Table II. In vitro activity of GST fusion poly(A) polymerase (GST–PAP) isolated from transfected NIH 3T3 cells.
| Proteina | λ phosphataseb | Relative PAP activityc |
|---|---|---|
| GST–PAP | – | 1.00 |
| GST–PAP | + | 1.08 ± 0.13 |
| GST–PAP + 14-3-3ε | – | 0.70 ± 0.02 |
| GST–PAP + 14-3-3ε | + | 0.90 ± 0.10 |
| GST–PAP + BSAd | – | 0.95 ± 0.14 |
| yPAP (positive control)e | – | 1.28 ± 0.08 |
| GST (negative control)f | – | 0.03 ± 0.00 |
aGST–PAP was purified by immobilization on glutathione–Sepharose beads from NIH 3T3 cells transfected with either both GST–PAP and HA-14-3-3ε constructs or GST–PAP alone. The polyadenylation reaction was carried out as in Figure 6B, and the reaction products were analyzed by the trichloroacetic acid precipitation assay.
bTreatment of the purified GST–PAP with λ phosphatase is indicated with +.
cThe relative PAP activity represents the amount of [α-32P]ATP incorporated by each protein fraction relative to that by GST–PAP without the λ phosphatase treatment.
dBSA of 2 µg was added in the reaction mixture.
eWe used 5 U of yeast PAP (yPAP; Invitrogen) as a positive control.
fWe purified the GST control protein from NIH 3T3 cells transfected with the GST host vector DNA as a negative control.