Figure 2.
ΔFosB is a phosphoprotein in brain. A, ΔFosB is phosphorylated in brain. Endogenous ΔFosB was induced in rat brain by chronic ECS treatment as described in Materials and Methods. Frontal cortical slices were metabolically labeled with 32P-H3PO4 for several hours. After homogenization of the slices, ΔFosB was immunoprecipitated, and phosphorylated ΔFosB (32P-ΔFosB) was detected by autoradiography (top panel). Total ΔFosB in nonradioactive immunoprecipitates was detected by immunoblotting (bottom panel). As negative controls, the immunoprecipitate of a sham-treated animal and nonimmune IgG are shown. B, Candidate phosphorylation sites and kinases with corresponding prediction scores for the mouse ΔFosB protein sequence were obtained by bioinformatic analysis. The candidate sites with the higher prediction scores are highlighted in the protein sequence and listed in the table. The DNA-binding (basic) domain and leucine zipper are in bold in the protein sequence. C, D, ΔFosB phosphorylation is increased by the Ser/Thr phosphatase inhibitor OA. C, 32P-ΔFosB levels in immunoprecipitates from frontal cortical slices labeled in the absence (Ctr) or presence of 100 ng/ml OA (graph and top panel) were detected by autoradiography. The bottom panel shows total ΔFosB detected in immunoprecipitates from unlabeled slices by immunoblotting. D, PC12 cells were infected with HSV-ΔFosB or HSV-LacZ (vector) and metabolically labeled with 32P-H3PO4 in the absence (Ctr) or presence of 100 ng/ml OA. 32P-ΔFosB levels in immunoprecipitates were detected by autoradiography (graph, top panel). The bottom panel shows total ΔFosB detected in cell lysates by immunoblotting.