Passive lysis buffer 1

The activity of the different
ligands was tested using a luciferase reporter gene assay. Receiver
cells stably expressing either Notch1 or synNotch variants were co-transfected
in a 24-well plate using TransIT-LT1 (Mirus) or Lipofectamine 3000
(Thermo Fisher Scientific, for U2OS cells) with a Gal4-firefly luciferase
reporter (Andrawes et al.^{32 (link)}) (300 ng) and
pRL-SV40 Renilla luciferase (10 ng). 24 h after transfection, the
cells were trypsinized and co-culture with cells stably expressing
the ligands. 48 h after plating, firefly luciferase and Renilla luciferase
activities were measured by luminometer (Veritas). Cells were lysed
with 100 μL/well passive lysis buffer 1× (Promega) for
10 min. 20 μL of each sample was used for luciferase activity
using filtered luciferase buffer including: 26 mg of (MgCO3)4 Mg(OH)2
(Sigma), 20 mM Tricine (Sigma), 0.1 mM EDTA (Biological Industries),
2.67 mM pH = 7.8 MgSO4 (Merck). For the luciferase reaction, we used
luciferase buffer supplemented with 0.4 mM ATP (Sigma), 26.6 mM DTT
(Sigma), Coenzyme A X0.8 (Sigma), and 0.4 mM d-Luciferin,
and for Renilla activity using filtered Renilla buffer including 80
mM di-potassium hydrogen phosphate trihydrate (Merck) and 20 mM potassium
dihydrogen phosphate for analysis (Merck). Notch activity is expressed
as a ratio of normalized luciferase by Renilla.

A total of 800,000 cells (in case of 96h transfection experiments) or 2 × 10⁵ cells (in the case of 24 h transfection experiments) were seeded into each well of a six-well plate and transfected with 0.5 μg of reporter constructs using Lipofectamine LTX (Invitrogen). For co-transfection experiments, 0.5 μg of expression plasmid or related empty vector were transfected in addition to the reporter constructs. The cells were lysed with Passive Lysis Buffer 1× (Promega, Mannheim, Germany) 24–96 h after transfection, and luciferase activity was determined. To normalize transfection efficiency, 0.2 μg of a pRL-TK plasmid (Promega, Mannheim, Germany) was co-transfected in each sample reaction and Renilla luciferase activity was measured by a luminometric assay (dual-luciferase reporter assay; Promega, Mannheim, Germany). Each experiment was repeated at least three times.