Figure 4

Gene-specific targeting of SHP-2 in C2C12 myotubes through vector-driven shRNA specifically knocks down SHP-2 protein. (A) C2C12 myotubes were transfected with pSUPER.shSHP2 construct targeting a specific region of the murine SHP-2 ORF. No transfection (Untransf.) or pSUPER vector only (pSUPER) transfected myotubes served as controls. Western blot analysis of SHP-2 levels revealed that pSUPER.shSHP2 knocked down > 60% of endogenous SHP-2. SHP-2 levels were quantitated by densitometric scanning and are shown as % of pSUPER vector only transfected myotubes (means ± SEM, from eight experiments). *** indicates a significant difference to the other bars (p < 0.0001; two-tailed unpaired t test). (B) The effect of SHP-2 gene targeting on levels of several postsynaptic proteins and functional surface AChRs was analyzed by pSUPER.shSHP2 transfection followed by Western blot analysis or radioligand binding assays (¹²⁵I-α-BTX binding to intact C2C12 myotubes). This revealed that SHP-2 knockdown does not significantly influence the levels of MuSK, Src, rapsyn and β-dystroglycan. Protein levels were calculated and shown as % of respective pSUPER vector only transfected myotubes (means ± SEM, from eight experiments). Equal amounts of overall protein were loaded on the gels. Similarly, ¹²⁵I-α-BTX binding to surface AChRs in intact C2C12 myotubes shows that following SHP-2 knockdown there is no significant effect on the surface levels of AChRs, when compared to radioligand binding on control transfected myotubes.