FIG. 5.

ECM does not induce transcriptional activation of nonintegrated templates in transient-transfection analysis. (A) Autoradiograph of an immunoprecipitation reaction using an antibody to mouse milk. The ³⁵S-labeled proteins were precipitated from parallel plates of transiently transfected cells cultured on plastic or low-density polyhema treated with 2% ECM. (B) A representative autoradiograph of a CAT assay. Ten micrograms of protein from cell lysates isolated from CID-9 cells transiently transfected with BBC or RSV/CAT while cultured on plastic (P) or low-density polyhema treated with 2% ECM (E) was analyzed. (C) A diagram for each plasmid tested in the transient-transfection analysis is shown to the left. BCE-1, β-casein, and MMTV are labeled relative to the transcription start site of the endogenous genes. The seven constructs contained the reporter CAT gene. The adjacent table represents the activity of each construct when transiently or stably transfected into CID-9 cells and differentiated in IHP on plastic or ECM as described in Materials and Methods. The transient activity is relative to cotransfected RSV–β-Gal expression (a promoter which is not regulated by the presence of ECM). −, no detectable activity; +, increasing amounts of transcriptional activity. These data represent at least three independent transfections for each condition.