Figure 3.

Results of Functional Analyses in CHO Cells

cDNAs encoding PGAP2 isoform 8 were subcloned into the vectors under promoters of different strengths and were expressed in PGAP2-deficient CHO cells.

(A) Immunoblot of cell lysates isolated with the strong SRα promoter after 2 days of expression showed similar levels of wild-type (lane 1) and altered (lane 2) PGAP2 proteins; an empty-vector construct did not express the protein (lane 3). The protein levels were normalized (levels are shown underneath the blot) with the intensities of GAPDH expression shown at the left part of the blots.

(B) Expression of the DAF and CD59 cell-surface proteins after transient transfection was monitored by FACS as a measurement of PGAP2 activities and is shown for the various promoter constructs. The left panel shows the p.Tyr99Cys alteration, and the right panel shows the p.Arg177Pro alteration. Under the strong SRα promoter, both altered proteins restore the cell-surface expression of DAF and CD59, suggesting a hypomorphic alteration. Expression under the weak promoters TK and TATA-box shows reduced activity of altered PGAP2 (see Results and Discussion for details).