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. 2019 Jul 25;4(14):e128643. doi: 10.1172/jci.insight.128643

Figure 7. Decreased stability of recombinant desmoplakin NH2-terminus with R451G mutation.

Figure 7

(A) A recombinant glutathione-S-transferase–tagged desmoplakin fragment consisting of the first 883 amino acids (GST-DSP1–883WT) was bacterially expressed and purified. After the fragment was incubated with calpain for 5-, 10-, 15-, or 30-minute intervals, degradation products were separated via SDS-PAGE and visualized by staining with Sypro Ruby. Very little degradation was observed. (B) The same experiment was repeated with a fragment containing the R451G amino acid substitution (GST-DSP1–883R451G). This gel shows substantial decrease of the intact fragment over time as it is exposed to calpain. (C) Quantification of gel images across repeated experiments reveals a significant increase in calpain-mediated degradation of the mutant R451G recombinant DSP protein relative to WT (*P < 0.05 for 2-tailed unpaired t test at 30-minute time point; GST-DSP1–883WT n = 4; GST-DSP1–883R451G n = 3). Error bars represent SEM.