IGF-1 does not significantly alter MDM2 protein stability or steady state mRNA levels.

<p>(A) U2-OS cells were serum-starved for 24 hours prior to treating with IGF-1 (50 ng/mL) for six hours. Cells were then treated with 50 µg/mL cycloheximide (CHX) in the presence or absence of IGF-1 and collected at the indicated times. Cell lysates were subjected to western blot analysis for MDM2 and actin. (B) Quantitative analyses of MDM2 protein levels from cells treated with CHX were performed by densitometry scanning for each individual MDM2 protein band and the corresponding actin protein band. The ratio of MDM2 over actin at the zero time point was arbitrarily set as 100 percent. Three independent experiments were performed with similar outcomes. (C) U2-OS cells were treated with IGF-1 (50 ng/mL) for six hours and metabolically labeled with ³⁵S-methionine for forty minutes, and then chased with cold methionine. Cell lysates with equal amounts of radioactivity were immunoprecipitated with SMP14 antibody against MDM2. Proteins were separated by SDS-PAGE and visualized by autoradiography. (D) Quantitative analysis was performed by densitometry scanning using ImageQuant software. (E) Serum-starved U2-OS cells were treated with IGF-1 (50 ng/mL) for six hours. MDM2 gene expression levels were determined by quantitative-PCR (Q-PCR) and normalized to GAPDH expression. Data presented as means and SE of two independent experiments performed in duplicate.