Figure 3
NFATp is a bona fide transcriptional activator. (A) Recombinant HA-NFATp activates transcription in an extract prepared from unstimulated Jurkat cell nuclei. The template used contained three NFAT sites upstream of the E1b TATA box and the CAT gene. Transcripts were analyzed by primer extension using a CAT specific primer followed by denaturing PAGE. The amounts of HA-NFATp used were: 12 ng and 40 ng. (B) Activation by HA-NFATp requires NFAT sites in the DNA template. Basal and NFATp-activated transcription were assessed using unstimulated Jurkat extract and either the (NFAT)3-E1b-CAT template or a template lacking NFAT sites (E1b-CAT). Transcripts were analyzed by primer extension using a CAT specific primer followed by denaturing PAGE. (C) Reconstitution of a purified RNA polymerase II transcription system that is devoid of AP-1 proteins. Transcription was reconstituted from highly purified factors and tested for response to cJun/cFos and cFos alone. The DNA template contained five high affinity AP-1 site upstream of the E1b TATA box and the CAT gene. Transcripts were analyzed by primer extension using a CAT specific primer followed by denaturing PAGE. (D) HA-NFATp activates transcription in a reconstituted transcription system. The DNA template was (NFAT)3-E1b-CAT. The transcription system consisted of highly purified recombinant and native general transcription factors as described in the text.