Tristar 5

The fluorescence of the supernatants was measured using a Tristar 5 plate reader (Berthold, Germany). Absorbance data were collected and processed using the MikroWin2010 program (Labsis Laborsysteme GmbH). The manufacturer's instructions were followed for operating the machine.

Fifty microliters of a 10 ng mL⁻¹ solution of a recombinant CypLase in 100 mM Tris–HCl (pH 7.5) containing 300 mM l-ascorbic acid sodium salt and 100 mM NaCl was automatically added to 50 μL of a 0.2 or 2 μM solution of chiral-separated (R)- or (S)-CypL in 100 mM Tris–HCl (pH 7.5) containing 300 mM l-ascorbic acid sodium salt and 100 mM NaCl on a black 96-well plate (FIA Plate; Greiner Bio-One, Kremsmunster, Austria) using an injector equipped with a multimode microplate reader (TriStar 5; Berthold Technologies, Bad Wildbad, Germany), followed by immediate measurement of luminescence intensity at room temperature. The luminescence intensity was recorded in relative light units (RLU) in 0.1 s intervals over 120 min.

The quadriceps were homogenized in 60 µL/mg ice-cold RIPA buffer (10 mM Tris–HCl pH 7.4, 5 mM EDTA, 0.1% SDS, 150 mM NaCl, 1% sodium deoxycholate, 1% Triton) with 1 mM phenylmethanesulfonyl fluoride and a protease/phosphatase inhibitor cocktail (#5872, Cell Signaling, Danvers, MA, USA) using a Bead Mill 4 Homogenizer (Thermo Fisher Scientific, Waltham, MA, USA), and then centrifuged (15,000× g for 10 min at 4 °C). The supernatant protein concentration was determined with Bradford Reagent (Sigma, Burlington, VT, USA) using a microplate reader (Tristar 5, Berthold, Bad Wildbad, Germany). Forty micrograms of denatured proteins were separated in SDS polyacrylamide 12% and transferred onto polyvinylidene fluoride membranes according to the manufacturer’s instructions (Invitrogen, Waltham, MA, USA). After overnight incubation with primary antibodies and then horseradish peroxidase-conjugated secondary antibodies (see Table S3 for details), the protein complexes were revealed (Kit Clarity Western ECL substrate, Bio-Rad, Hercules, CA, USA) using chemiluminescence (Fusion X Spectra, Vilber, Marne-la-Vallée, France). The protein expression was quantified with ImageJ software (NIH, Bethesda, MD, USA, https://imagej.nih.gov/ij/) and normalized to glyceraldehyde 3-phosphate dehydrogenase (GADPH).