Inhibitors of the FGFr-diacylglycerol pathway inhibit NCAM and FGF-induced increased GAP-43 phosphorylation
| Control | NCAM | FGF | |
|---|---|---|---|
| Control | 47.2 ± 3.5 | 98.2 ± 4.0* | 60.4 ± 7.1** |
| αFGFR | 44.1 ± 4.2 | 63.9 ± 3.1*** | 47.7 ± 4.5 |
| 766 | 42.1 ± 3.8 | 74.4 ± 2.69*** | 45.9 ± 8 |
| 766P | 36.4 ± 2.6 | 39.2 ± 3.4 | 35.9 ± 4.7 |
| RHC-80267 | 42.6 ± 6.8 | 38.4 ± 5.9 | 37.5 ± 5.0 |
IGS prepared from P2 mice forebrains were treated at 37°C with either Krebs’s buffer alone or containing αFGFR antiserum, used at 1:200; the peptide 766 and its phosphorylated form 766P, used at 5 μg/ml; or the diacylglygerol lipase inhibitor RHC-80267, used at 10 μg/ml. After 30 min the samples were centrifuged and resuspended in either buffer alone or buffer containing NCAM-Fc, used at 5 μg/ml or FGF2, used at 25 ng/ml. After 15 min the samples quickly were frozen in liquid nitrogen. Equal amounts of total protein (5 μg) were slot-blotted in triplicate, and parallel blots were reacted with the mAb 2G12 to detect phosphorylated GAP-43 or with 7B10 to detect total GAP-43. Immunoreactivity was detected with an iodinated secondary antibody and quantitated by a phosphorimager. The results show the specific activity of phosphorylated GAP-43 and are the mean of three independent experiments ± SEM. Specific activities were compared with controls, using two-tailed Student’s t test.
*p < 0.001; **p > 0.05; ***p > 0.01.