Exogenous JunB does not overcome inhibition of monopoiesis by C/EBPα.
(A) Total cellular proteins prepared from 1 × 10632DPKCδ-αER-MTJunB-1 or -2 cells cultured under the indicated conditions for 16 hours were subjected to Western blotting for JunB and β-actin. (B) Total cellular proteins from 32DPKCδ-αER-MTcJun-1 and -2 cells were analyzed similarly for c-Jun and β-actin. (C) Total cellular RNAs, 10 μg per sample, prepared from αER-MTJunB-2 cells cultured under the indicated conditions for 16 hours were subjected to Northern blotting for macrosialin (MS) and β-actin. (D) Nuclear extracts prepared from αER-MTJunB-2 cells cultured with no inducer, PMA, PMA + estradiol, or PMA + estradiol + zinc for 8 hours were subjected to gel-shift analysis using a radiolabeled AP-1 binding site derived from the macrosialin promoter. (E) Total cellular protein derived from 1 × 106 αER-MTJunB-2 cells exposed to no inducer (−), PMA + zinc for 4 hours (PZ), PMA + zinc followed by cycloheximide for 15 minutes (CHX), or the latter cells exposed to estradiol or the vehicle control for 30, 60, 90, or 120 minutes were subjected to Western blot analysis for JunB (top panel). As a control for protein content, each extract was electrophoresed on a second gel and visualized with Coomassie blue dye (bottom panel).
