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Figure 6


Fig. 6. SH2-Bß is involved in GDNF-induced neuronal differentiation of PC12-GFR{alpha}1-RET cells. (A) PC12-GFR{alpha}1-RET cells were co-transfected with pEGFP-N2 (encoding GFP) in the ratio of 1:10 with pcDNA3 as control, pcDNA3-myc-SH2-Bß or R555E. The cells were visualized using fluorescence microscopy (magnification 20x) based on the expression of the GFP. (B) Quantification of neuronal differentiation of PC12-GFR{alpha}1-RET cells. Values are the mean ± s.d. of three independent experiments performed in triplicate culture wells. Three fields were examined from each well. *, significant difference between SH2-Bß/GFP and pcDNA3/GFP control (P<0.05, factorial ANOVA); ##, significant difference between R555E/GFP and control (P<0.01, factorial ANOVA). (C) Decrease in GDNF-induced differentiation of PC12-GFR{alpha}1-RET cells by SH2-Bß-siRNA. PC12-GFR{alpha}1-RET cells cultured on 24-well plates were transfected with pEGFP-N2 alone or together with missense RNA (20 µM) as control, or with SH2-Bß-siRNA (20 µM). (D) Quantification of neuronal differentiation of PC12-GFR{alpha}1-RET cells. Values are the mean ± s.d. of three independent experiments. **, significant difference between RNAi group and missense RNA group (P<0.01, factorial ANOVA). (E) Effect of overexpression or knock-down of SH2-Bß on GDNF-induced activation of ERK and Akt in PC12-GFR{alpha}1-RET cells. PC12-GFR{alpha}1-RET cells were transfected with plasmids pcDNA3 (control), pcDNA3-myc-SH2-Bß, pcDNA3-myc-R555E, missense RNA or SH2-Bß-siRNA. Cells were stimulated with or without 100 ng/ml GDNF for 10 minutes and then lysed. Equal amounts of total protein of cell lysates were immunoblotted with anti-SH2-Bß, anti-pERK, anti-ERK, anti-pAkt or anti-Akt antibody.





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