Figure 8

Time course study of c-Myc and phospho-extracellular signal regulated kinase (ERK)1/2 expression by western blot analysis. Serum-deprived nucleus pulposus cells were treated with or without 16 μM 10058-F4 or 30 μM PD98059 before the addition of 5 ng/mL transforming growth factor β1 (TGFβ1). The cells were harvested at the times indicated and lysed. Aliquots of the lysates were electrophoresed on 5% to 20% gradient sodium dodecyl sulfate polyacrylamide gel electrophoresis SDS-PAGE). The protein bands were blotted to a polyvinylidene diflouride (PVDF) membrane and probed with antibodies against c-Myc, total ERK1/2 and phospho-ERK1/2. β-Actin was used as a quantity loading control. (a) TGFβ1 treatment induced immediate phosphorylation of ERK1/2 with robust c-Myc expression for 2 h. The expression of c-Myc, phospho-ERK1/2, and total ERK1/2 were detected throughout the experimental period. The right lane indicates the result of 24 h treatment with 10% FBS; c-Myc and phospho-ERK1/2 appear distinctly. (b) Pretreatment with ERK1/2 inhibitor 30 μM PD98059 diminished the expression of c-Myc and interrupted the phosphorylation of ERK1/2. Note that a single isoform corresponding to phospho-ERK2 was detected at all times. (c) Pretreatment with c-Myc inhibitor 16 μM 10058-F4 diminished c-Myc expression and limited ERK1/2 phosphorylation for a short time under TGFβ1 stimulation. Graphs show relative intensities in expression of c-Myc normalized to β-actin levels and in expression of phospho-ERK1/2 normalized to total ERK1/2 levels, respectively.