ATP assays were performed as previously described [29 (link)]. In short, age-matched wild-type and mutant larvae were collected and homogenized in ATP extraction buffer (100 mM Tris-Cl, pH 8.0, 4 mM EDTA, pH 8.0, and 6 M guanidine hydrochloride) and used for both ATP and bicinchoninic acid (BCA) assay. ATP concentrations were determined using ATP determination Kit (Invitrogen, Thermo Fischer Scientific, Waltham, MA, USA) as per the manufacturer’s protocol. The luciferase activity was measured in 96-well format using a CLARIOstar plate reader (BMG Labtech Inc., Cary, NC, USA). Protein concentrations were determined using a BCA assay. 5 µL of diluted samples (1:50) were mixed with 100 µL of BCA reagent (Pierce BCA protein assay Kit, Thermo Fischer Scientific, Waltham, MA, USA), and protein concentrations were measured on a CLARIOstar plate reader (BMG Labtech Inc., Cary, NC, USA). Results were averaged over three (Rswl) or five (Scu) technical replicates and represented as ATP concentrations normalized to the protein concentrations.
Free full text: Click here
