Figure 6
Analysis of the scFv expression in eukaryotic cells. Panel A. Western blot analysis of extracts from the HPV16-positive SiHa cells transfected with plasmids expressing the scFv 32, 43M2 and 51, targeted to the cellular cytoplasm (lane c), nucleus (lane n) and endoplasmic reticulum (lane er). The analysis was performed 48 hours after transfection. Non transfected cell lysates were used as negative control. The Western blotting was performed using the rabbit anti-c-myc mAb (clone 9E10) followed by GAM-HRP IgG incubation and chemiluminescence. The scFvs are indicated by arrows. To control for protein loading, the blots were probed with a mouse anti-actin mAb (Sigma). Panel B. Analysis of the scFv solubility. Cos-7 cells were transfected with plasmids expressing in the cell cytoplasm the scFv 43 (43), 43M2 (M2), 32, 51 and the highly stable and soluble R4 anti-β-gal scFv as a control. The cellular extracts were fractionated in soluble (lanes s) and insoluble (lanes i) fractions. The presence of the antibodies in these fractions was examined by Western blotting performed as above described.