96 well microtiter plate

The activity of the 26S proteasome was measured^{47 (link)} by following steps. Cells were lysed in a cytosolic extract buffer on ice and were centrifuged at 10,000×g for 15 min at 4 °C. Four to eight micrograms of total protein was diluted with 26S proteasome assay buffer in a 96-well microtiter plate (BD Falcon), and incubated with fluorogenic substrate. Suc-LLVY-AMC, Ac-nLPnLD-AMC, and Ac-RLR-AMC (Enzo) were used to measure chymotrypsin-, caspase-, and trypsin-like proteasome activity, respectively. Fluorescence released by AMC fluorescence was monitored on a microplate fluorometer (Infinite M200, Tecan) every 5 min at 37 °C for 1 h. The specificity of the assay was assessed using the proteins inhibitors bortezomib (BTZ, 0.2 μM) and carfilzomib (CFZ, 0.5 μM), the latter being an effective inhibitor for the trypsin- and caspase-like activity.

S. aureus (AR0215) and A. baumannii (AR0083) (see Table S2, Supporting Information) were grown overnight (stationary phase) prior to diluting in 10 mL LB broth. For the bactericidal kinetics studies using S. aureus, the LB broth was supplemented with either chloramphenicol or ceftazidime at 1 x MIC (Table S3, Supporting Information). For A. baumannii bactericidal studies, the LB broth was supplemented with either chloramphenicol or vancomycin. Bacterial suspensions were initially plated for CFU as above, and subsequently exposed to either 18 J cm⁻² aBL (A. baumannii) or 108 J cm⁻² (S. aureus), prior to incubating at 37 °C. Bacteria were then quantified every hour for a total of 8 h in order to gauge viability over time. For the inhibition studies, bacteria were exposed to the same antibiotics as described above. Bacteria were exposed to either 18, 36, 54, 108, or 216 J cm⁻², reflecting 5, 10, 15, 30, or 60 min of aBL, prior to transferring 200 µL of the bacteria/LB/antibiotic mix to a 96‐well microtiter plate (Falcon, USA) and measuring the OD₆₀₀ every 30 min for a total of 14 h.

Worms were lysed in proteasome activity assay buffer (50 mM Tris-HCl, pH 7.5, 10% glycerol, 0.5 mM EDTA, 5 mM MgCl₂, 2 mM ATP and 1 mM dithiothreitol) using a Precellys 24 homogenizer (Bertin technologies). Human cells were collected in proteasome activity assay buffer and lysed by passing 10 times through a 27 G needle attached to a 1 ml syringe. C. elegans and human cell lysates were centrifuged at 10,000 × g for 10 min at 4 °C. For each sample, 25 μg of total protein were transferred to a 96-well microtiter plate (BD Falcon) and incubated with fluorogenic proteasome substrates. To measure trypsin-like proteasome activity, we used Ac-Arg-Leu-Arg-AMC (Enzo, BWL-AW9785-0005). For chymotrypsin-like and caspase-like proteasome activities, we used Z-Gly-Gly-Leu-AMC (Enzo, BML-ZW8505-0005) and Z-Leu-Leu-Glu-AMC (Enzo, BWL-ZW9345-0005), respectively. Fluorescence accumulation upon proteasomal degradation of the fluorogenic substrate (380 nm excitation, 460 nm emission) was measured on a microplate fluorometer (EnSpire, Perkin Elmer) every 5 min for 1 h. Then, the slope of fluorescence accumulation over time was calculated. To average independent replicate experiments and perform statistical analysis, we normalized the slope from the test conditions to the respective control condition of the same replicate experiment.