A. 10–20% Tris-tricine Coomassie-stained SDS-PAGE gel showing peptides generated using alkylated liver bSOD1. Lanes 1, 6 and 12 are molecular weight standards; Lane 2, undigested bSOD1; Lane 3, endoproteinase Lys-C (Lys-C) only; Lane 4, Lys-C digestion; Lane 5, Lys-C and thrombin only; Lane 7, Lys-C + thrombin digestion; Lane 8, Lys-C and glutamyl endopeptidase (Glu-C) only; Lane 9, Lys-C + Glu-C digestion; Lane 10, Lys-C, Glu-C and thrombin only; Lane 11, Lys-C + Glu-C + thrombin digested bSOD1. Arrows indicate a peptide that is present in undigested SOD1 and retained in SOD1 digested to completion with endoproteinase Lys-C, thrombin and glutamyl endopeptidase as outlined.
B. Activity assay using either untreated bSOD1, sequentially and completely digested protein or a protease only control as cofactor material. “Triple Digestion” corresponds to lane 11 in (A).
C. Western blot analysis of bSOD1 from a 1:1000 proteinase K: bSOD1 (w/w) digestion over a 120 min time course. Anti-SOD1 (1:5000) was used as the primary antibody in this analysis.
D. Specific phospholipase activity (nmol PED6 cleaved/min/mg rExoU) when proteinase K digested products from denatured bSOD1 are used to activate rExoU over a digestion time course (error bars ± SEM, n = 3). Untreated refers to bSOD1 that is not denatured nor protease treated and undigested refers to bSOD1 that is denatured but not treated with proteinase K.