Fig. 5. Complex formation with DNA and Chip stabilizes Ap protein. (A) Immunoblot of S2 cell lysates transfected with constructs to express myc-tagged Ap and myc-tagged dLMO. Cells were transfected with a constant amount of Ap-myc (+) and increasing amounts of dLMO-myc as indicated. Total levels of transfected DNA were held constant using empty vector to compensate for alteration in the level of dLMO-myc plasmid. Both proteins were visualized with anti-myc. The arrowhead indicates a nonspecific band to serve as a loading control. (B) Immunoblot of S2 cell lysates transfected with a plasmid to express myc-tagged Ap and with plasmids containing additional Ap-binding sites. Cells were transfected with a constant amount of Ap-myc (+) and increasing amounts of plasmid containing additional Ap-binding sites (competitor). Total DNA levels were held constant in the transfection by addition of empty vector. Ap protein levels increased with increasing copies of the binding site construct. (C) Immunoblot of S2 cell lysates transfected with constructs to express myc-tagged Ap and myc-tagged ChAp. Cells were transfected with a constant amount of Ap-myc (+) and increasing amounts of ChAp-myc as indicated. Both proteins were visualized with anti-myc. Increasing amounts of ChAp decreased levels of Ap-myc protein. We tested whether the endogenous dLMO gene was induced in ChAp transfected cells by immunoblotting with anti-dLMO, and were unable to detect it (not shown). The level of dLMO that was needed to reduce Ap-myc protein in S2 cells was readily detectable by immunoblotting, so we conclude that the effect of ChAp-myc was not due to increased levels of dLMO.
