Cell cultures were grown as in the monoculture growth assay for fitness and then fractionated following a previously described procedure (Mehlhoff et al. 2020 (link)) based on the BugBuster reagent protocol for cell fractionation. Further description of measurement of the resulting protein concentration by DC Assay Kit (Bio-Rad) and sample preparation for SDS-PAGE can be found there as well. We loaded 15 μg of protein from fractionated samples in each lane for SDS-PAGE gels. Protein fractions were analyzed on 12% NuPAGE Bis-Tris gels (Thermo Fisher) in MOPS running buffer. Gels were stained using Coomassie Brilliant Blue R-250 (Bio-Rad) to verify even loading of lanes. We performed immunoblots using rabbit Anti-CAT-I antibody (C9336; Sigma), rabbit Anti-NDM-1 antibody (MBS715336; MyBioSource), and rabbit Anti-AadB antibody (Cusabio custom antibody). All western blot results were substantiated by biological replicates (supplementary figs. S9, S11, and S12, Supplementary Material online).
Since CAT-I is a homotrimer in its native form (Biswas et al. 2012 (link)), we used Native-PAGE and Western blots to see if the quaternary structure of CAT-I was affected by select mutations found to have deleterious collateral fitness effects. Samples for Native-PAGE were prepared using 15 μg of protein, 4X NativePAGE sample buffer (Invitrogen), 0.25 μl NativePAGE 5% G-250 Sample Additive, and nuclease-free water before being loaded onto a NativePAGE 4–16% Bis-Tris Gel (Thermo Fisher). The inner chamber of the electrophoresis system was filled with 1X NativePAGE Dark Blue Cathode Buffer (Thermo Fisher) while the outer chamber was filled with 1X NativePAGE Anode Buffer (Thermo Fisher). The gel was run for approximately two hours at 150 V with the 1X NativePAGE Dark Blue Cathode Buffer being replaced with 1X NativePAGE Light Blue Cathode Buffer (Thermo Fisher) after the dye front had migrated one third of the way through the gel. We then proceeded with Western blots as described.
Since CAT-I is a homotrimer in its native form (Biswas et al. 2012 (link)), we used Native-PAGE and Western blots to see if the quaternary structure of CAT-I was affected by select mutations found to have deleterious collateral fitness effects. Samples for Native-PAGE were prepared using 15 μg of protein, 4X NativePAGE sample buffer (Invitrogen), 0.25 μl NativePAGE 5% G-250 Sample Additive, and nuclease-free water before being loaded onto a NativePAGE 4–16% Bis-Tris Gel (Thermo Fisher). The inner chamber of the electrophoresis system was filled with 1X NativePAGE Dark Blue Cathode Buffer (Thermo Fisher) while the outer chamber was filled with 1X NativePAGE Anode Buffer (Thermo Fisher). The gel was run for approximately two hours at 150 V with the 1X NativePAGE Dark Blue Cathode Buffer being replaced with 1X NativePAGE Light Blue Cathode Buffer (Thermo Fisher) after the dye front had migrated one third of the way through the gel. We then proceeded with Western blots as described.
