Effects of luteolin on IGF-I-induced tyrosine phosphorylation of IGF-IR, the association of p85 with IGF-IR, and PI3K activity in human colon cancer cells. Cells were plated and cultured as described in Figure 1. (A) HT-29 cells were treated for 2 h with 0 or 60 μmol/L of luteolin and lysed with or without stimulation of 10 nmol/L IGF-I for 0, 1, or 30 minutes. Total cell lysates were incubated with anti-IGF-IRβ antibody and the immune complexes were precipitated with protein A-Sepharose. The immunoprecipitated proteins were analyzed via Western blotting with antibodies raised against phosphotyrosine (PY20), IGF-IRβ, or p85. (B) HT-29 and Caco-2 cells were plated and treated as described above. Total cell lysates were analyzed via Western blotting with an antibody raised against P-IGF-IR. Photographs of the chemiluminescent detection of the blots, which were representative of three independent experiments, were shown. (C) The immune complexes obtained from HT-29 cells were incubated with phosphatidylinositol and [γ-32P]ATP. (D) Active PI3K and luteolin were incubated with phosphatidylinositol and [γ-32P]ATP as described in the Materials and Methods section. Phosphatidylinositol 3-phosphate (PIP) generated by immunoprecipitated PI3K (C) or active PI3Kα (D) was separated via thin-layer chromatography (TLC). An autoradiograph of the TLC plate, which was representative of three independent experiments, is shown. (A, B, C) The relative abundance of each blot was quantified via densitometric scanning of the exposed films and the control levels (0 μmol/L luteolin, without IGF-I stimulation) were set at 100%. Each bar represents the mean ± SEM (n = 3). *Different from 0 μmol/L of luteolin at a stimulation time, P < 0.05.