FIG. 10.
Half-lives of SfIAPM1 and leaderless SfIAPM4. (A) SfIAP expression vector. The T7 epitope-tagged SfIAP gene was placed under the control of the ie-1 promoter linked to the AcMNPV hr5 enhancer. mRNA transcription initiates within the promoter (+1) and terminates at the polyadenylation site (PA). Translation begins at the AUG codon within the N-terminal T7 tag. Thus, the translational context of each construct is identical. (B) Protein stability assays. SF21 cells transfected 24 h earlier with plasmid encoding the indicated SfIAPs were treated with 400 μg cycloheximide (CHX) per ml. Cell lysates prepared at the indicated times thereafter were serially diluted and analyzed by immunoblotting with anti-T7 and anti-actin. A representative blot is shown; constant actin levels demonstrated comparable protein loading. Half-life values were calculated from SfIAP signal intensity on films exposed in the linear range. Values shown are the averages ± standard deviations of protein half-lives (in minutes) derived from three independent experiments.