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Figure 6


Fig. 6. Inhibition of FGF-mediated PC12 neurite formation by overexpression of APRO4. PC12 cells were transfected with either a control empty vector (data not shown), Flag-APRO4 (A), X-APRO4{Delta}N (B), Flag-APRO4{Delta}C (C), or Flag-APRO4 and HA-MEK1 S218/222D (D), stimulated with FGF (25 ng/ml) for 2 days, fixed and immunostained with the indicated antibodies. FGF-stimulated transfected PC12 cells expressing Flag-APRO4 (panel A, white arrows) or X-APRO4{Delta}N (panel B, white arrows) did not extend neurites whereas Flag-APRO4{Delta}C transfected PC12 cells did (panel C, white arrows). Untransfected PC12 cells stimulated with FGF developed neurites (panels A-C, black arrows). In cells co-transfected with Flag-APRO4 and HA-MEK1 S218/222D, cells expressing only Flag-APRO4 (red) did not extend neurites (panel D, white arrows) whereas cells expressing Flag-APRO4 (red) as well as HA-MEK1 S218/222D (green) expression plasmids showed extensive neurite outgrowth (panel D, black arrows). Polyclonal anti-Flag antibody, monoclonal anti-Xpress and anti-HA antibodies showed no signal with untransfected PC12 cells (panels A-C, black arrows). More than 300 transfected PC12 cells were analysed per condition in four independent experiments. Bars, 10 µm.





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