PU.1 and AP-1 DNA-binding and PU.1 activity in 32DPKCδ-C/EBPα-ER cells.

(A) Nuclear extracts prepared from 32DPKCδ-αER-1 cells cultured in PMA, estradiol (E2), both, or neither for 8 hours were subjected to gel-shift assay (lanes 1-3, 10) using as probe 1 ng of a radio-labeled PU.l-binding site derived from the macrosialin promoter. Also added to the gel-shift reactions as indicated were 50, 100, or 200 ng wild-type (W) or mutant (M) probe, 2 μL PU.1 antiserum (Ab), or 2 μL normal rabbit serum (RS). Asterisks (*) denote faster migrating species that likely represent proteolytic fragments of PU.1. (B) To assess PU.1 or C/EBP activities, 20 μg (PU.1)₄TKLUC, (mPU.1)₄TKLUC, or (C/EBP)₂TKLUC and 2 μg pCMV-βGal were transfected into 2.0 × 10⁷ 32DPKCδ-αER cells, which were then split and cultured ± 4HT or in PMA ± 4HT, as indicated. Luciferase and β-galactosidase activities were determined 24 hours later and used to calculate a normalized luciferase activity. Ratios of these activities in the presence versus the absence of 4HT are shown ([activity in 4HT or 4HT + PMA]/[activity in no inducer or PMA]) for each DNA construct (mean and SE of 2 determinations). (C) The same extracts described in panel A were subjected to gel-shift assay using as probe an AP-1 binding site from the macrosialin promoter (lanes 1, 4, 7, and 10). Included as an unlabeled competitor in the gel-shift reactions when indicated were 50-fold excess of wild-type or mutant probe.