Figure 2

Mitogen-activated protein kinase signaling in response to dynamic strain (DS). Articular chondrocytes (ACs) were exposed to no treatment or to treatment with interleukin-1-beta (IL-1β), DS alone, or DS and IL-1β for 10 or 30 minutes and were examined by Western blot analysis for (a) Ser217/221-MEK1/2 phosphorylation by DS and IL-1β. Equal protein loading was confirmed by probing blots with anti-total MEK1/2 antibody. (b) Ser338-c-RAF phosphorylation, (c) Ser445-B-Raf phosphorylation, and (d) Ras activation following immunoprecipitation with GST-Raf-1-RBD and glutathione agarose beads are shown. (e) Ras activation following pretreatment of cells with a selective Ras antagonist (2 μM GGT12133) for 30 minutes is shown along with the assessment of Ras-dependent phospho-Thr202/Tyr204-ERK1/2 (P-ERK1/2) 10 and 30 minutes post-activation. Anti-total ERK1/2 IgG (T-ERK1/2) was used to normalize total input in all lanes. All experiments were performed in triplicate. Gels represent one of three experiments with similar results in (a-e). The graphs above gels in each figure show mean and standard error of the mean of phosphoprotein/total protein in three separate experiments in (A-D). *P < 0.05 as compared with untreated control cells; **P < 0.05 as compared with IL-1β-treated cells. DTF, dynamic tensile force; ERK1/2, extracellular receptor kinase 1/2; GGT, Ras inhibitor GGT12133; GST, glutathione-S-transferase; MEK1/2, mitogen-activated protein kinase/extracellular receptor kinase 1/2; RBD, Ras-binding domain.