Fig. 3. Binding of wild-type but not mutant Ad9 E4-ORF1 to endogenous ZO-2 of CREF cells. One milligram of protein from RIPA buffer-lysed normal CREF cells or CREF cells stably expressing wild-type or mutant Ad9 E4-ORF1 were immunoprecipitated with ZO-2 antiserum or the matched pre-immune serum (pre). Recovered proteins were separated by SDS–PAGE and immunoblotted with either ZO-2 or Ad9 E4-ORF1 antiserum (top panel). As a control, 100 µg of protein from RIPA buffer-lysed normal CREF cells was also directly immunoblotted with the same antisera (bottom panel). Sample 1 of the top panel was not analyzed in the bottom panel.