Fig. 2. c-fos induction in cultured cortical neurons by C-Ag4,8 is concentration dependent and saturable.a, Cortical cultures were treated with C-Ag4,8 or control medium in NBM for 10 min at room temperature, returned to cNBM, and incubated for 2 hr at 37°C after which Fos levels were determined using the quantitative assay described in Materials and Methods. Levels of Fos expression, shown in arbitrary OD405 units, increased in a C-Ag4,8concentration–dependent manner (filled circles). Basal levels of Fos expression (open circles) were low and did not change significantly with an increasing concentration of control medium added to the culture. The graph shows Ab-2–specific binding; nonspecific binding, determined by labeling C-Ag4,8– or control medium–treated cultures with Ab-2 preadsorbed to an excess of peptide antigen, was <5% of total binding and has been subtracted. The C-Ag4,8 concentration is given in AU where one unit is the amount of C-Ag4,8 required to induce a half-maximal increase in AChR clusters assayed on cultured chick myotubes. Each point represents the mean ± SEM for triplicate determinations. Similar results were seen in two other experiments. b, To test the specificity of C-Ag4,8 induction of Fos expression, we immunoprecipitated an aliquot of C-Ag4,8–containing medium with the anti-agrin monoclonal antibody Agr 530. The chart showsc-fos–inducing (open bars; left scale) or AChR-clustering (shaded bars;right scale) activity measured in cortical neuron cultures and cultured chick myotubes, respectively, after treatment with a saturating concentration of C-Ag4,8medium (C-Ag4,8) or an equivalent amount of C-Ag4,8 medium immunoprecipitated in the absence (−530) or presence (+530) of Agr 530. Greater than 90% of thec-fos–inducing and AChR-clustering activities are removed from the C-Ag4,8 medium by immunoprecipitation with Agr 530, confirming that C-Ag4,8 is responsible forc-fos induction in cortical neurons. Basal levels of Fos observed in cortical cultures treated with sham-conditioned medium and spontaneous AChR clusters have been subtracted. Error bars represent the mean ± SEM for triplicate determinations. Similar results were also seen in a second experiment.