Fig. 5.

CMT1C-associated mutations reduce the stability of SIMPLE protein in cells. (A) HeLa cells co-transfected with pEGFP vector and an equal amount of cDNAs encoding Myc-tagged WT, W116G, or P135T were analyzed by immunoblotting with antibodies against the Myc tag, β-actin and GFP. (B,C) Quantification of protein levels from A. After normalization to βactin (B) or GFP (C), SIMPLE W116G and P135T protein levels relative to that of SIMPLE WT are shown as mean ± s.e.m. from three independent experiments. *P<0.05 compared with SIMPLE WT. (D) The degradation of Myc-tagged SIMPLE WT, W116G or P135T proteins expressed in HEK293 cells were analyzed by [³⁵S]Met/Cys pulse-chase assays. ³⁵S-labeled SIMPLE WT or mutant proteins were immunoprecipitated from lysates with anti-Myc antibodies and detected by autoradiography. (E) The protein levels of WT and mutant SIMPLE from D were quantified and plotted relative to the corresponding protein levels at 0 hours. Data represent mean ± s.e.m. from three independent experiments.