Fig. 2. FGF2 treatment leads to serine but not tyrosine phosphorylation of STAT in RCS chondrocytes. (A,B) RCS chondrocytes were serum-starved for 12 hours, treated with FGF2, IL6 or IFN
for the indicated times, and analyzed for ERK1/2, STAT1, STAT3 and STAT5 by WB with antibody recognizing the given molecule only when phosphorylated at a specific site. The arrow indicates Y694-phosphorylated STAT5. The same membrane was re-probed with antibody recognizing the given molecule regardless of its phosphorylation. (C) RCS chondrocytes were serum-starved for 12 hours, treated with FGF2 (100 ng/ml) in the presence of heparin (1 µg/ml) for the indicated times, and analyzed for STAT3 by WB with antibody recognizing STAT3 only when phosphorylated at S727. The appearance of S727-phosphorylated STAT3, triggered by FGF2, is indicated by an arrow. The lower band appears to be a crossreactivity of the antibody as indicated by the position of the total STAT3 detected on the re-probed membrane.