Fig. 5

HSP90 inhibitors, geldanamycin and onalespib, stimulate the phosphorylation of SAPK/JNK induced by TGF-β in osteoblastic MC3T3-E1 cells. The cultured osteoblasts were pretreated with 0.3, 0.7, 1.0 μM of geldanamycin (A) and 0.3, μM of onalespib (B) for 60 min, and then incubated by 3 ng/ml of TGF-β or vehicle for 2 h. Cell extracts were analyzed by SDS-PAGE and Western blotting using antibodies of phospho-specific SAPK/JNK or SAPK/JNK. The histogram shows the quantitative representations of the levels of phosphorylated SAPK/JNK normalized with each total SAPK/JNK gained from laser densitometric analysis. The levels were expressed as the fold increase to the basal levels presented as lane 1. Triplicate determinations of Western blot analysis were performed corresponding to three independent cell preparations. Each value represents the mean ± S.E.M. of triplicate determinations from three independent cell preparations. *P < 0.05, compared to the value of the control cells without TGF-β-stimulation. **P < 0.05, compared to the value of TGF-β alone