In vitro transcription from the V
BCL1 promoter is stimulated by the IgH enhancer core alone (Eμ) or the enhancer and associated MARs (Eμ+MARs) on templates reconstituted under Step 1 conditions. Transcription reactions were carried out as described in Materials and Methods on ~50 ng templates in which either the VHBCL1 promoter alone (P-only), or the Eμ core without (Eμ) or with its flanking MARs (Eμ+MARs), were positioned ~2 kbp 3'. Transcription was initiated by addition of NTPs. Upper panel: BCL1 transcripts, which initiate at two major sites in vivo, were detected as described in Fig. 1C by RNase protection following fractionation on denatured gels. Lane 1, ~2 μg yeast RNA; lane 2, ~2 μg total RNA from BCL1 leukemia cells; N, naked DNA; D, pre-binding with BJAB nuclear extract (~5 μg/reaction) prior to assembly of chromatin (Step 1 conditions of Fig. 1E); R, post-binding of BJAB extract following assembly of chromatin (Step 2 conditions). Lower panel: Corresponding western blot of SDS-PAGE-fractionated reactions (~15 μg/lane) to monitor and normalize Bright protein levels within input BJAB nuclear extract.