Figure 4.2-C. Evaluation of freshly isolated normal human bronchial epithe- 
lial cells for the expression of adenovirus protein following infection 
with AvlCFl. The expression of adenovirus genes after infection of human 
bronchial epithelial cells by the recombinant adenovirus AvlCFl was exam- 
ined at the level of protein expression by evaluating for the presence of 
adenovirus Ad hexon protein with and without immunoprecipitation. Human 
bronchial epithelial cefls were obtained at bronchoscopy using a standard 
cytology brush as previously described (Trapnell et al., 1991a) and were 
cultured in suspension in Dulbecco's modified Eagle medium, 10% fetal calf 
serum (FBS) , 1% glutamine, 100 U/ml penicillin, and 100 /zg/ml streptomycin, 
and infected with AvlCFl at a multiplicity of infection (MOI) of 100 plaque 
forming units (pfu) per cell in medium as above except the FBS was 2% . 
After 90 minutes , FBS was brought to 10% and the cells were cultured for 3 
days. The cells were washed twice with cold PBS and labeled with [ 35 S] 
methionine (1000 Ci/mmole, 500 /iCi/ml) for 24 hr. Following the labeling 
period, cells were washed in PBS, and lysed in buffer containing 
antiproteases (see Appendix 1 for details). The cell lysate was cleared of 
debris by brief (10 min) centrifugation in a microfuge. The cleared cell 
lysate was subjected to sodium dodecyl sulfate acrylamide gel electropho- 
resis and autoradiography. Cleared lysate was also subjected to 
immunoprecipitation using an anti-Ad5 hexon antibody and protein A. Ad 
hexon protein was not seen in uninfected cells in the absence (lane 1) or 
presence of immunoprecipitation (lane 4) , but was abundantly expressed in 
cells infected by Ad5 with (lane 2) and without (lane 5) 
immunoprecipitation. Ad hexon protein could not be detected after AvlCFl 
infection of cells either without (lane 3) or with immunoprecipitation 
(lane 6). As further controls, while Ad hexon could not be detected in 
uninfected 293 cells (lane 7), hexon was easily detected after only 1 day 
after infection by either Ad5 (lane 8) or AvlCFl (lane 9). 
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Recombinant DNA Research, Volume 16 
