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. 2011 Dec 1;7(12):e1002396. doi: 10.1371/journal.pgen.1002396

Figure 10. The Ogna mutation sensitizes plectin's RD to degradation by epidermis-specific proteolytic activities.

Figure 10

(A) Aliquots (10 µg) of GST-tagged wild-type RD (Figure 6A) were incubated (30 min, 30°C) with (+) or without (−) epidermal protein extract (5 µg) in the presence of the protease inhibitors indicated [MDL, MDL-28170 (50 µM); ALLN, N-Acetyl-Leu-Leu-Nle-CHO (5 µM); MG, MG132 (Z-Leu-Leu-Leu-al, 20 µM); EDTA (10 mM), PMSF (2 mM)], or vehicle (DMSO) alone. Samples were separated by SDS-8% PAGE and RDs were detected by immunoblotting using anti-plectin mAb 10F6. Note that RDs exhibit electrophoretic mobilities slightly lower than expected for a protein with a calculated molecular mass of ∼158 kDa. Also note multiple RD degradation products (*) in lanes DMSO and MG. (B) Assay conditions as in (A), except that 10 or 100 nM Bortezomib (BTZ), and 0.9% (w/v) NaCl were used as protease inhibitor and sole vehicle, respectively. (C) Degradation of wild-type (WT) and mutant plectin RDs (O) by epidermal protease(s) as a function of incubation time. Assay conditions as in (A), except that no protease inhibitors were added and incubations times varied as indicated. Note faster degradation of the Ogna compared to the wild-type RD version. Arrows in (A–C) denote the position of intact RD proteins. Note faster degradation of the Ogna RD compared to the wild-type rod version. (D) Degradation kinetics of wild-type versus Ogna RD. The relative amounts of intact RD (as determined by densitometry) were plotted as a function of incubation time with epidermal protein extract. Data are shown as mean ±SEM of three independent experiments.