Fig. 5. C/EBPß expression, binding activity and localization in 3T3-L1 transfectants. (A) Control and 1-PDX cells were subjected to an adipogenic treatment as described in Materials and Methods. Cell extracts were prepared on days 0, 1, 2, 3, 5 and 7. Aliquots of 25 µl were fractionated by SDS-PAGE and analyzed by immunoblotting for C/EBPß. The antibody recognized the LAP (liver-enriched activating protein, 32 kDa) and the LIP (liver-enriched inhibitor protein, 18 kDa) C/EBPß isoforms. Control and 1-PDX producing cells carried equivalent amounts of both isoforms. (B) Nuclear extracts were prepared from control or 1-PDX expressing preadipocytes treated with adipogenesis-inducing agents for 24 hours. An EMSA of a radiolabeled C/EBP consensus oligonucleotide probe was conducted using 10 µg of nuclear extract proteins. For supershift assays, the extract was preincubated with an antibody directed against the C-terminus of C/EBPß. In competition assays, a 100-fold molar excess of unlabeled oligonucleotide was supplemented to the binding mixture. (C) The nuclear extracts (50 µg of proteins) analyzed for binding activity in (B) were fractionated by SDS-PAGE and further analyzed by immunoblotting for C/EBPß. Extracts from 1-PDX-expressing cells contained less C/EBPß LAP than those from control cells (lanes 1,2). The upper band in lane 1 corresponded to a hyperphosphorylated form of C/EBPß as demonstrated by its disappearance in CIAP-treated extracts (lanes 3,4). The blots shown are representative of three separate experiments with three control and three 1-PDX clonal lines. (D) Immunohistochemical analysis on 3T3-L1 transfectant cells after adipogenic treatment for 24 hours. Localization of C/EBPß was nuclear in control cells (c) and perinuclear in 1-PDX-expressing cells (d). Panels a and c represent control samples not treated with the anti-C/EBPß primary antibody.