Spectramax plus 284

The kinetics of the FsC reaction
on PET were measured by using a suspension-based assay originally
described by Arnling Bååth et al.,^{17 (link)} at three different pH values (5.0, 6.5, and 9.0). Reactions were
set up in triplicate in Eppendorf tubes with a total volume of 600
μL, containing 10 g L^–1 crystalline PET powder
(GoodFellow product code ES306031; 37.7 ± 2.6% crystallinity^{20 (link)}), enzyme concentrations varying between 0–1
μM, and either a 25 mM sodium acetate buffer, pH 5.0, a 25 mM
sodium phosphate buffer, pH 6.5, containing 50 mM NaCl, or a 50 mM
glycine buffer, pH 9.0.
The reactions were incubated in an Eppendorf
ThermoMixer C at 40 °C and 450 rpm for 7 h. At 0, 1, 3, 5, and
7 h 100 μL of was transferred from each reaction to a 96-well
MultiScreen_HTS HV Filter Plate (0.45 μm pore size;
Millipore), and the reactions were stopped by vacuum filtering using
a Vac-Man 96 Vaccum Manifold (Promega) onto a 96-well Clear Flat Bottom
UV-Transparent Microplate (Corning). PET hydrolysis products were
quantified by measuring A₂₄₀ in a Spectramax Plus 284 microplate
reader (Molecular Devices), and concentrations were calculated by
using a standard curve made with 15, 30, 60, 90, 120, and 150 μM
TPA (Figure S2).

NF-κB was determined in nuclear extracts from gastrocnemius muscle according to a modified protocol by Dignam et al.^{63 (link)} using an ELISA-based TransAM NF-κB p65 assay kit (Active Motif, Carlsbad, CA, USA) in accordance with the manufacturer’s instructions. Sample absorbance was read at 450 nm on a multiwell microplate reader (Spectra Max plus 284, Molecular Devices). Wild-type and mutated consensus oligonucleotides were used as competitors for NF-κB binding to ensure specificity of the reaction as per the manufacturer’s instructions. All the tests of the samples were run in duplicate, and the average value was used for data analysis.