Luciferase cell lysis buffer

Gluc activity was measured by adding 50 μl Gluc assay reagent (Nanolight Technology, Pinetop, AZ, USA) to 20 μl of the Gluc sample and analyzing with a GLOMAX 96 microporous plate light reader (Promega, Madison, WI, USA) to determine the bioluminescence. Fluc activity was similarly analyzed: 20 μl luciferase cell lysis buffer (NEB, Ipswich, MA, USA) was added to cellular extract, and then 100 μl of the Fluc assay reagent (Lulong, Xiamen, China) was added to the cell lysate and read immediately.

A Spry1 promoter region (–341 to +39 bp relative to the transcription start site [TSS]) and a Pdk3 promoter region (–107 to +101 bp relative to the TSS) were PCR amplified from genomic DNA (C57BL/6 male) and subcloned into pGL4.18 (Promega). pGL4.18_Atp7a has been reported previously (35 (link)). pGL4.18-Spry1, pGL4.18-Atp7a, and pGL4.18-Pdk3 (200 ng each) were cotransfected with pcDNA3-HIF1ATM, pcDNA3-HIF2ATM, or a control empty pcDNA3 plasmid (200 ng) into C2C12 myoblasts. pcDNA-LacZ (100 ng) expressing β-gal was cotransfected as an internal transfection control. Forty-eight hours after transfection, myoblasts were lysed with Luciferase Cell Lysis Buffer (NEB), followed by a firefly luciferase/β-gal assay with homemade luciferase and β-gal solutions. The luciferase assay solution consisted of 0.11 M Trizma, pH7.8, 0.5 M MgCl₂, 0.1 M ATP, and 10 mM luciferin. The β-gal solution consisted of 4 mg/ml O-nitrophenyl-β-D-galactoside, 60 mM Na₂HPO₄, 40 mM NaH₂PO₄, 10 mM KCl, and 1 mM MgSO₄.

ODD-Luc mice were infected and euthanized as described above, and lungs and spleens were collected. Lungs were homogenized with DPBS and protease inhibitor (Roche), and an aliquot was reserved for luciferase quantification. Luciferase cell lysis buffer (New England Biolabs, Ipswich, MA) was added to form a homogenate, and the reaction mixture was incubated at room temperature for 15 min. Protein concentrations were quantified using the bicinchoninic acid (BCA) assay (Thermo, Fisher). Thirty micrograms of protein was added to an opaque Corning 96-well plate (Corning, NY), and luciferase buffer was added using a BioTek Synergy multimode plate reader (BioTek). Luciferase buffer was composed of 4.8 ml 0.11 mM Tris (pH 7.8), 50 µl 100 mM sodium luciferin, 60 µl 200 mM ATP, and 120 µl 0.5 M MgCl₂.

Total cell extracts equivalent to 1 × 10⁶ cells were separated on SDS-polyacrylamide gels and subject to either standard or LICOR western blotting analysis according to the manufacturers’ instructions. For standard western blots, duplicate gels were generated and one was stained with Coomassie and the other was used to produce the nitrocellulose blot. Blots were blocked in 5% milk in TBST and washes were performed in TBST (0.05% Tween). Blots were then probed with 1/1000 α-gLUC primary antibody (New England Biolabs) and 1/2000 α-rabbit secondary antibody (Bio-Rad). For LICOR blots, blocking was performed in 50 mM Tris, pH 7.4, 0.15 M NaCl, 0.25% BSA, 0.05% Tween, 2% fish skin gelatine (Sigma, UK) and washes were performed in TBST (α-gLUC) or PBST (α-GFP). Nitrocellulose blots were incubated with 1/1000 α-gLUC or 1/5000 α-GFP (Life Technologies) and 1/20 α-tubulin (kind gift from Keith Gull), followed by 1/10,000 α-mouse and 1/10,000 α-rabbit IR Dye antibodies (LICOR). Lysates for gLUC assays were prepared by adding 20 µl of 1 × luciferase cell lysis buffer (New England Biolabs) to 2 × 10⁶ pelleted cells and these were directly used to perform BioLux Gaussia luciferase assays (New England Biolabs) according to the manufacturers’ instructions and using a TopCount plate-reader with white-walled plates. One-way ANOVA tests were carried out in GraphPad Prism (version 7).