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. 2003 Jun;23(12):4066–4082. doi: 10.1128/MCB.23.12.4066-4082.2003

FIG. 7.

FIG. 7.

Decreased C/EBP-β phosphorylation in cGMP-treated cells. (A) C6 cells were transfected with an expression vector encoding C/EBP-β and either empty vector (left panel, lanes 1 to 5), G-kinase II (right panel, lanes 1 to 5) or a G-kinase I/II chimera (right panel, lanes 6 and 7); cells were incubated with 32PO4 for 4 h and treated for 1 h with buffer (lanes 1, 5, and 6), 0.3 μM A23187 (lane 2), 250 μM CPT-cGMP (lane 3), or both agents (lanes 4 and 7) as described in Materials and Methods. Cell lysates were subjected to immunoprecipitation with a rabbit anti-C/EBP-β antibody (lanes 1 to 4, 6, and 7) or control rabbit IgG (lane 5). Immunoprecipitates were analyzed by SDS-PAGE, electroblotting, and autoradiography (upper panel) and by blotting with a murine anti-C/EBP-β antibody (lower panel). The immunoglobulin heavy chain band is labeled Ig. (B) 32PO4 incorporation into C/EBP-β was quantitated by scanning densitometry of autoradiographs of three independent experiments performed as described for panel A; cells were transfected with C/EBP-β plus either empty vector (open bars) or G-kinase II (solid bars). (C) Cells were transfected with C/EBP-β and G-kinase II and labeled for 4 h in 32PO4-containing medium as described for panel A. Half of the cultures were treated with 0.3 μM A23187 and 250 μM CPT-cGMP for the indicated times, and 32PO4 incorporation into C/EBP-β was quantitated as described for panel B. Data are expressed as percentages of the level in untreated controls. (D) Cells transfected with C/EBP-β were labeled with 32PO4 for 4 h and either left untreated (lanes 1 and 2) or treated for 1 h with 5 mM sodium valproate (lane 3); cell lysates were processed as described for panel A (lane 1, control IgG; lanes 2 and 3, anti-C/EBP-β antibody). (E) Cells were transfected with pCRE-Luc, pRSV-βGal, and either empty vector (E.V.) or 12 ng of C/EBP-β vector; cultures were either left untreated (open bars) or treated with 5 mM sodium valproate for 8 h (solid bars). Luciferase activity was normalized to β-galactosidase activity as described for Fig. 3.