Figure 6

FGF1 phosphorylation inhibits its anti-apoptotic activity in SH-SY5Y cells. SH-SY5Y cells were stably transfected with FGF1^WT, FGF1^K132E, FGF1^S130A, FGF^S130D or empty (mock) expression vectors. (a) FGF1 levels were examined by western blot after heparin-sepharose concentration in cell lysates and conditioned media from SH-SY5Y cells expressing FGF1^WT, FGF1^S130A, FGF1^S130D or transfected with an empty vector (mock). (b) Apoptosis in FGF1^WT, FGF1^K132E, FGF1^S130A, FGF^S130D or mock stably transfected SH-SY5Y cells after 48 h of etoposide treatment was analyzed by flow cytometry after DiOC₆(3) and PI staining. Apoptotic cells are the DIOC−, PI− and size− cells. The graph represents the mean±S.E.M. of three independent experiments. Student’s t-tests were performed relative to the control mock, except where indicated (n=3; n.s.: P>0.05; *: P⩽0.05; ***: P⩽0.001). (c,d) PUMA, cleaved caspase-3 and PARP (full-length and cleaved forms) levels were analyzed by western blot in FGF1^WT, FGF1^K132E, FGF1^S130A, FGF^S130D or mock stably transfected SH-SY5Y cells after 0, 4, 8 or 16 h of etoposide treatment. Actin detection was used as control