Tyrphostin AG1296 (Tyrphostin AG 1296) is an inhibitor of PDGFR with IC50 of 0.3-0.5 μM, no activity to EGFR.

PDGFR:0.3 μM-0.5 μM, FGFR (Swiss 3T3):12.3 μM, c-Kit (Swiss 3T3):1.8 μM

AG 1296 选择性地抑制 PDGF 受体激酶以及在 Swiss 3T3 细胞和猪主动脉内皮细胞中的 PDGF 依赖的 DNA 合成，其50%抑制浓度分别低于 5 和 1μM。此外，AG1296 在 Swiss 3T3 细胞中对 FGFR 和 c-Kit 的 IC50 分别为 12.3 μM 和 1.8 μM。AG1296 强效抑制人 PDGF-α 和 -β 受体的信号传导，但对 VEGFR KDR 的自磷酸化或猪主动脉内皮细胞中由 VEGF 引起的 DNA 合成无影响。AG1296 的处理可以逆转 sis-转染的 NIH 3T3 细胞的转化表型，但对 src-转化的 NIH3T3 细胞无效。AG1296 是一种 ATP-竞争性抑制剂，不干扰 PDGF 的结合或 PDGF 受体二聚化，而是消除 PDGF 受体的自磷酸化，因此，AG1296 是一种纯粹的受体酪氨酸激酶的催化活性抑制剂。

Membrane Autophosphorylation Assays:Membranes are prepared from confluent cultures of Swiss 3T3 cells as described. For measuring receptor autophosphorylation, 10μg membrane protein per assay are incubated for 20 min on ice in the presence of 1.2μg/mL EGF or 2μg/mL PDGF, or both; 50 mM Hepes (pH 7.5); and 3 mM MnCl2 in a volume of 45μl. In order to test the effects of tyrphostins, these are added in a volume of 0.5 μl (in DMSO; final concentration, 0.5%) 15 min before addition of the growth factors. Phosphorylation is initiated by addition of [γ-32P]ATP and terminated after 2 min by addition of 10μL of a solution containing 6% SDS, 30%β-mercatoethanol, 40% glycerol, and 0.5 mg/mL bromophenol blue. The samples are heated for 5 min at 95 ℃ and subjected to SDS-PAGE using 10% acrylamide gels. The gels are stained and dried and subjected to autoradiographic analysis.

Cell lines: Swiss 3T3. Concentrations: ~50 μM. Incubation Time: 3 days. Method: Cells are seeded in 24-well plates (5000 cells/well) in DMEM/10% FCS.On the next day the medium is changed to DMEM/2% FcS with or without growth factors and tyrphostins are added as indicated.Three days later the cells are counted in a hemocytometer