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. 2014 Aug 14;9(8):e104583. doi: 10.1371/journal.pone.0104583

Figure 3. Disulfide crosslinking of mutant SOD1 contributes to electrophoretic heterogeneity.

Figure 3

HEK293FT cells were transiently transfected with expression vectors for WT and mutant SOD1. After 24 hours, soluble extracts of the cells were prepared by lysis in digitonin, analyzed by BNGE, and immunoblotted with the antibody identified on the figure. (A) A portion of the cell lysate was treated with 2-mercaptoethanol (final concentration 1%) before electrophoresis. Gels containing samples treated with 2-mercaptoethanol (BME) were transferred separately from gels containing untreated samples. (B and C) A portion of the cell lysate was analyzed without BME treatment and immunoblotted with either antibody to SOD1 or antibody to ubiquitin. After transfer, the membranes were air dried to fix proteins to the membrane and then incubated with primary and secondary antibodies in parallel, with all three membranes imaged simultaneously. Treatment with 2-mercaptoethanol reduced amount of, but did not eliminate, slowly migrating G93A SOD1. A mutant form of SOD1 lacking all 4 cysteines produced low levels of slowly migrating forms of SOD1 immunoreactivity than the G93A mutant. The images shown are representative of at least 3 independent replications.