Tyrphostin 23 (AG18) inhibits EGFR with IC50 of 35 μM.

EGFR:35 μM

AG18以剂量依赖性方式抑制原代星形胶质细胞培养物中[3H]牛磺酸的体积敏感释放。AG 18以剂量依赖性方式抑制原代星形胶质细胞培养物中[3H]牛磺酸的体积敏感释放。 AG 18在A431细胞中抑制EGF诱导的EGFR自体磷酸化,IC50为15 μM。10 μM AG 18抑制EGF诱导的GH3细胞增殖。10 μM AG 18抑制GH3细胞中Ghrelin刺激的ERK1/2磷酸化增加。10 μM AG 18转移2倍KCl诱导的收缩,且产生最大的抑制作用。100 μM AG 18抑制A549上皮细胞中TNF-alpha和TPA刺激的IKK活性。300 μM AG 18剂量依赖性抑制A549上皮细胞中TPA诱导的ICAM-1表达刺激。300 μM AG 18作用于A549 上皮细胞,也抑制TPA刺激的NF-kappaB DNA-蛋白结合和ICAM-1启动子活性。

EGF-Receptor Autophosphorylation: WGA-purified EGF receptor from A431 cells (0.5 μg/assay) is activated with EGF (800 nM) for 20 min at 4 ℃. The reaction is initiated by the addition of Mg(Ac)2 (60 mM), Tris-Mes buffer, pH 7.6 (50 mM), and [32P]ATP (20 pM, 5 μCi/assay). The reaction is conducted at either 4 ℃ or 15 ℃ and terminated by addition of sodium dodecyl sulfate (SDS) sample buffer (10% glycerol, 50 mM Tris, pH 6.8, 5% β-mercaptoethanol, and 3% SDS). The samples are run on a 8% SDS polyacrylamide gel (SDS-PAGE) (prepared from 30% acrylamide and 0.8% bis-(acrylamide) and contained 0.375 M Tris, pH 8.8, 0.1% SDS, 0.05% TEMED, and 0.46% ammonium persulfate). The gel is dried and autoradiography is perfromed with Agfa Curix RP2 X-ray film. The relevant radioactive bands are cut and counted in the Cerenkov mode. The fast phase of autophosphorylation continued for another 10 min. The extent of phosphorylation completed in the first 10 s at 15 ℃ comprises 1/3 of the total autophosphorylation signal and probably reflects the phosphorylation of the first site on the receptor. The 10-s interval is therefore chosen for use in subsequent autophosphorylation experiments.

GH3 cells are plated at 5 × 104 cells/well in media containing 2% charcoal-stripped FCS and various concentrations of ghrelin, desoctanoylated ghrelin and PMA or EGF for 72 hours with the addition of 2 μCi/well [3H]thymidine for a further 6 hours. A time-course of 24 hours, 48 hours and 72 hours is performed for ghrelin stimulation and 72 hours is selected for further experiments. Studies are also performed to investigate the effect of rat ghrelin or desoctanoyl ghrelin-induced proliferation and the effect of U0126, GF109203X, AG 18, wortmannin and H-89 upon ghrelin-induced MAPK stimulation. AG 18 at 10 μM is added 30 min before each treatment. Cells are harvested before counting in the presence of scintillation fluid using a Microbeta 1450 bcounter. Experiments are repeated at least three times.(Only for Reference)