Figure 2.

BMN 111 Reduced ERK1/2 Activation in ACH Growth-Plate Chondrocytes

(A) Control and pathologic human chondrocytes (ACH) were isolated as described previously²⁴ and treated with BMN 111 (10⁻⁵ M) prior to coincubation with fibroblast growth factor (FGF) (100 ng/ml FGF18). BMN 111 pretreatment partially prevented FGF-mediated increase in ERK1/2 phosphorylation (n = 6). The cell lysates were subjected to SDS polyacrylamide-gel electrophoresis and were hybridized overnight at 4°C with phosphorylated-ERK1/2 antibody (1/1,000) (Cell Signaling Technology, Danvers, MA, USA) or ERK1/2 antibody (1/1,000) (Cell Signaling Technology). A secondary antibody coupled to peroxidase was used at a dilution of 1:10,000 (GE Healthcare Life Sciences, Pittsburgh, PA, USA). Bound proteins were detected by enhanced chemiluminescence (GE Healthcare Life Sciences).

(B) Immunoblot showing STAT3-P and STAT3 (dilution 1:1,000, Cell Signaling Technology) in human ACH growth-plate chondrocytes (n = 3). The phosphorylation of STAT3 did not appear decreased upon BMN 111 coincubation.