Fig. 5.

BT assays of in vitro-expressed mutant (89I) and wild-type Dnas1l3. Dnase1l3 protein levels as assessed in lysates of HeLa cells transfected with nuclease-expressing plasmids. (a) Representative lysates used to condition media show approximately twofold higher levels of the mutant protein by anti-Dnase1l3 immunoblot. Actin levels were nearly equivalent (data not shown). (b) Representative RDAs of conditioned media show near equivalent FD-nuclease activity. The twofold higher level of 89I expression appears to equalize RDA activity. (c) However, when the respective conditioned media are added to naive HeLa and assayed for the presence of BT activity, media with wild-type 89T enzyme provides a nearly eightfold greater barrier to liposomal transfection of GFP. (d) Graphic demonstration of the results of anti-GF Western blots performed for BT assays. As depicted, media conditioned with wild-type enzyme blocks transfection significantly more than media conditioned with 89I Dnase1l3.