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. 2024 Aug 1;9(8):e00089-24. doi: 10.1128/msphere.00089-24

Fig 4.

Gel images depict protein band intensity at different antibiotic concentrations for strains with mutations compared to the control. Bar charts display relative bocillin intensity and the effect of pre-incubation with amoxicillin on protein binding.

AMX-Bocillin competitive binding assay. The relative signal intensity of Bocillin was set to maximal when no AMX was added to the binding assay reaction (AMX preincubation concentration = 0). However, preincubation with 0.0156–0.031 µg/mL of AMX showed a higher Bocillin signal in transformant 26695_Fr5-8 (A, C) than transformant 26695_Fr3-8 (B, D), indicating a decrease in PBP1A affinity for AMX in transformants carrying mutations within PBP1A, with a greater effect seen with PBP1A harboring four mutations than PBP1A harboring only two mutations. The total protein intensity for each lane (not shown) was used for Bocillin quantification. Three independent assays were performed (n = 3).