Luciferase and β-Galactosidase Assays

Cells were lysed by the addition of 100 μl of passive lysis buffer (Promega/Biotium) to each well and shaken for >15min. Luciferase assay was carried out as described in (Chan and Egan, 2005 (link); Chan and Egan, 2009 (link); Egan et al., 2013 (link); Chan et al., 2021 (link)) in a buffer containing 0.0165 M glycylglycine, 0.01 M MgSO₄, 2.65 mM EGTA, 10.5 mM potassium phosphate, 1.4 mM adenosine 5′-triphosphate, 0.86 mM dithiothreitol (DTT), 0.175 mg/ml bovine serum albumin (BSA), and 0.035 mM luciferin (Promega) using 50 μl of the lysate and a luminometer (Berthold Technologies, Germany). A standard curve was constructed using recombinant luciferase (Promega). Beta-galactosidase assay was carried out as described in (Chan and Egan, 2005 (link); Chan and Egan, 2009 (link); Egan et al., 2013 (link)) in a buffer containing 100 mM sodium phosphate pH7.3, 1mM MgCl₂, 50 mM β-mercaptoethanol and 0.665 mg/ml o-nitrophenyl β-D-galactopyranoside (Sigma) using 20 μl of the lysate and read at 420 nm using a plate reader (Bio-Tek Synergy HT). Luciferase activity was normalized against β-galactosidase activity.