Saponification analysis of exported GspB736flag and GspB1060flag.
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A) GspB736flag and GspB1060flag secreted from parental strains PS1225 (lane 1) and PS921 (lane 3) and the corresponding derivative strains harboring the S362A mutation within asp2, PS3539 (lane 2), PS3540 (lane 4) were separated by SDS-PAGE and subjected to Western blot analysis using anti-FLAG antibodies to detect GspB levels. Saponified reaction products were obtained through incubation with 100 mM NaOH to release ester-linked acetate (lanes 5–8). B) Blot A was simultaneously probed for GlcNAc reactivity of GspB736flag and GspB1060flag before and after saponfication. GlcNAc reactivity was assessed by lectin blot analysis using biotinylated sWGA as a GlcNAc probe. Western and lectin blot analysis of secreted GspB variants are representative of 3 different genetic transformants analyzed from each strain.