Fig. 7. hFBNRG3 is exported to the plasma membrane and released to the extracellular medium. (A) hFBNRG3 is post-translationally modified with N-acetyl-galactosamine but not with N-acetyl-glucosamine as demonstrated by the absence of affinity for the WGA lectin (lanes 2 and 3) and its binding to SBA lectin (lanes 4 and 5). Nonspecific binding was ruled out by elution with galactose (lane 6 and 7). (B) Dose-response curve of erbB4 phosphorylation. COS-7 cells transiently transfected with pcDNA3-erbB4 were trypsinized and re-seeded to assure identical levels of erbB4 receptor expression per well; after serum deprivation, cells were incubated with the indicated amount of recombinant NRG3 (inset) for 5 minutes, harvested with sample buffer and western blotted with anti-phosphotyrosine antibody. Increases in phosphotyrosine levels were normalized after re-probing the membranes with anti-PKC-. The results of three different experiments are given as the mean + s.e.m. (top). (C) The same approach was used to detect neuregulin activity in conditioned medium (CM) of hFBNRG3 transfected cells. As shown, erbB4 phosphorylating activity was found in the medium of MG-132-treated, transiently transfected cells (lane 6). Although in some experiments neuregulin activity in the medium of nontreated cells was detected, differences were not statistically significant (lane 4 and bar labelled CM). Basal phosphorylation of erbB4 was estimated from conditioned medium of cells transfected with the inversely oriented hFBNRG3 cDNA without (lane 3) or with (lane 5) MG-132 and also in GST-treated cells (lane 7), and subtracted from calculations. Positive phosphorylation control was performed by adding recombinant GST-NRG3-EGF-like protein (lane 8). No protein bands were detected in nontransfected COS-7 cells even when GST or recombinant neuregulin was added (lanes 1 or 2, respectively). Data are given as mean ± s.e.m (n=3, ^*P0.05); Student's t-test was used.