Supporting Information

Files in this Data Supplement:

Fig. 7. The 14-3-3 g protein did not affect the agrin-induced acetylcholine receptor (AChR) clustering in C2C12 myotubes. (a) Three- to four-day-old myotubes were incubated with Torpedo agrin (10 ng/ml) for 16 h at 37°C. AChR clusters (Top) were detected with FITC-conjugated a -bungarotoxin (1 m g/ml, Molecular Probes). The 14-3-3 g protein (Middle) was detected with anti-14-3-3 g polyclonal antibody after permeabilization with 0.5% Triton X-100. Merged images (Bottom) illustrated the absence of 14-3-3 g immunoreactivity from agrin-elicited AChR clusters in C2C12 myotubes. (b) Myotubes overexpressing 14-3-3 g (identified by means of cotransfected GFP, Lower) exhibited normal AChR clusters (Upper). Quantitative analysis of data in b are shown in the histogram (n = 8). (Scale bar, 10 m m.).

Fig. 8. The 14-3-3 g protein represses synaptic genes transcription by the mitogen-activated protein kinase and phosphatidylinositol-3 kinase (PI3K) pathways in C2C12 myotubes. C2C12 cells were transfected with constitutively active Ras (Ras V12), Raf (Rafcaax) or PI3K (p110*) in addition to e -AChR reporter gene and 14-3-3 g construct. Ras V12, Rafcaax, and p110* strongly activated the expression of the e -AChR reporter gene. The overexpression of the 14-3-3 g protein almost totally reversed these effects. Experimental conditions were similar to Fig. 4. Constitutively active Ras (Ras V12/acRas) and PI3K (acPI3K) were generous gifts from A. Klippel (Atugen, Berlin) (Hu, Q., Klippel, A., Muslin, A. J., Fanti, W. J. & Williams, L. T. (1995) Science 268, 100–102.). Rafcaax, a constitutively active Raf, was purchased from Clontech.