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. 2011 Oct 1;124(19):3319–3331. doi: 10.1242/jcs.087114

Fig. 6.

Fig. 6.

CMT1C-associated mutations promote aggregation of SIMPLE. (A) Stably transfected HEK293 cells expressing Myc-tagged WT, W116G or P135T SIMPLE were treated for 24 hours with either 2 μM MG132 or vehicle control (CTRL). Cell lysates (T) were separated into TX-100-soluble (S) and -insoluble (I) fractions. Aliquots representing an equal percentage of each fraction were analyzed by immunoblotting with antibodies against SIMPLE and β-actin. (B) The relative level of WT or mutant SIMPLE proteins in the insoluble fraction was quantified and expressed as a percentage of the total SIMPLE protein level in the corresponding cell lysate. Data represent mean ± s.e.m. from three independent experiments. *P<0.05 compared with SIMPLE WT under the same treatment. (C) HEK293 cells expressing GFP or GFP-tagged WT or mutant SIMPLE were treated for 2 hours with 2 μM dithiobis(succinimidylpropionate) (DSP) or vehicle control (CTRL) and analyzed by immunoblotting with an anti-GFP antibody. Asterisk indicates cross-linked protein complexes formed between SIMPLE and its interacting proteins.