Nano glo hibit extracellular detection system

CHO cells were seeded in 96-well plates and transiently transfected with pBiT3.1-C-PiT-2 (WT, D28N, G120R, A227V, C496Y, or mock). Translocation rates of PiT-2 were measured using the Nano-Glo HiBiT Extracellular Detection System according to the manufacturer’s protocol (Promega). Briefly, HiBiT is a small tag protein of 11 amino acids that binds to LgBiT, and the HiBiT–LgBiT complex emits luminescence upon substrate binding. Because LgBiT is impermeable to the cell membrane, only the HiBiT–LgBiT complex on the cell membrane surface is detected. The membrane translocation rate was calculated as the ratio of the membrane protein expression level to the total protein expression level. The transfection efficiency of each vector was confirmed via the co-transfection of GFP. Nano-Glo HiBiT Extracellular Reagent was prepared in advance and added to the transfected CHO cells. Cells were incubated for 10 min, and luminescence of the membrane protein was measured using the GloMax Multi Detection System (Promega). After measurement, cells were lysed with Triton-X100, and total protein was measured.

For quantifying HiBiT-tagged ADAM17 expression on the cell surface, the Nano-Glo HiBiT Extracellular Detection System (Promega, Madison, WI, USA) and Nano-Glo HiBiT Lytic Detection System (Promega) were used according to the manufacturer’s instructions^{18 (link)}. Adam10/17^−/− mEFs transfected with pHiBiT-ADAM17-WT, pHiBiT-ADAM17-C567R, or pHiBiT-ADAM17-C600Y for 48 h in 96-well microplates were incubated with PMA for 5 min and washed with PBS. After subsequent incubation at 37 °C in DMEM for 2 h, the cell culture medium in each well was replaced by 100 μL of Nano-Glo HiBiT Extracellular Reagent, and the plate was incubated at 25 °C for 10 min (Supplementary Figure S2b). Luciferase activity was measured using a SpectraMax i3x microplate reader (Molecular Devices). Then, cells were washed with PBS three times and 100 μL of Nano-Glo HiBiT Lytic Reagent was added to the well. After incubation at 25 °C for 10 min, luciferase activity was measured in each sample. The ratio of extracellular and lytic luminescence intensity for each well was calculated.

HEK293T cells were plated on white 96-well plates (Corning, NY, USA) with 25,000 cells per well and allowed to grow in complete medium. Cells were then transfected with 400 ng human AT₁Rs tagged with HiBiT cloned into pcDNA3. Four hours after transfection, the medium was changed to complete medium. The cells were then incubated in starvation medium for a further day. On the day of stimulation, HEK293T cells were incubated for different durations (10 min, 3 and 6 h) and stimulated with 1 µM Ang II or 1 mg/mL AT₁R-Abs (dialyzed against DMEM Low Glucose for 24 h).
Alternatively, HEK293T cells were pre-incubated with AT₁R-Abs (1 mg/mL) or DMEM low glucose for six hours, then stimulated with different concentrations of Ang II (10 nM, 100 nM, or 1 µM) for 10 min.
In every case, cells were treated following stimulation with the Nano-Glo HiBiT extracellular detection system (Promega Corporation, Madison, WI, USA) according to the manufacturer’s instructions.

Huh-7 cells were seeded at a density of 1.3 × 10⁴ in white 96-well microplates (Thermo Fisher Scientific). The next day, cells were transfected with plasmids encoding for HiBiT-M_N3Q, HiBiT-M_N3Q-∆20_ct, HiBiT-M_N3Q-₂₁₁DxE₂₁₃A and HiBiT-M_N3Q-₁₉₉KxGxYR₂₀₄A at a concentration of 100 ng/well using the TransIT®-LT1 Transfection Reagent. Sixteen hours post-transfection, the medium was removed and 50 µl DMEM without FCS was added to each well. To assess the plasma membrane expression, a mix containing 50 µl extracellular buffer, 0.5 µl LgBiT, and 1 µl extracellular substrate was added per well (Nano-Glo® HiBiT Extracellular Detection System-Promega). The total protein expression levels were assessed by adding a mix containing 50 µl lytic buffer, 0.5 µl LgBiT, and 1 µl lytic substrate (Nano-Glo® HiBiT Lytic Detection System-Promega). Luciferase activity was measured by the use of a Tristar LB941 luminometer (Berthold Technologies). For each construct, the ratio plasma membrane signal/total protein signal was calculated and plotted relative to the wild-type M. For each condition, duplicate wells were taken and experiments were repeated at least three times.

IFNλs were produced using the protocol described previously, which is capable of generating functional IFNλs (17 (link)). Briefly, IFN expression plasmids were transfected into sub-confluent HEK-293T cell monolayers, which are hyporesponsive to IFNλ signalling due to very low expression of IFNλR1 (15 (link)). Lipofectamine 2000 was used to transfect IFNλ plasmids per manufacturer’s instructions. IFNλs were routinely generated in six-well plates or 10 cm dishes, and 2 and 14 µg of plasmids were used, respectively. Lipofectamine 2000 (2 µl) was used per microgram of plasmid. Plasmids were transfected into cells in Optimem for 16–18 hours, before changing media to growth media (10% FCS) until 2 days posttransfection was reached. The conditioned media were harvested, clarified by centrifugation, aliquoted, and immediately frozen at −80 in. Relative levels of IFNλs were estimated using the extracellular HiBiT split luciferase assay by virtue of their C-terminal HiBiT tag by incubating IFN preparations with assay reagents and measured by manufacturer’s instructions (Nano-Glo HiBiT Extracellular Detection system, Promega) using a luminometer.

Internalization of the β₂AR was performed using the Nano-Glo® HiBiT Extracellular Detection System from Promega. For this purpose, the human β₂AR was provided with an N-terminal 11 amino acids long tag (HiBiT, VSGWRLFKKIS) using PCR technology. HEK293 cells were transfected with the HiBiT-β₂AR DNA construct by polyethyleneimine (PEI) method. Briefly, cells were seeded in 12 well plates and allowed to grow until a confluency of ~80 % was reached. Before transfection medium was changed to 900 μl fresh medium. Two μg DNA were diluted in 100 μl 150 mM NaCl. 6.6 μl of a PEI stock solution (10 mg PEI in 10 ml ddH2O) were added. After an incubation of 10 min at room temperature, 100 μl of the mixture was added to the cells. Culture vessel was centrifuged for 5 min at 280 g. After 24 h, medium was changed to fresh DMEM containing antibiotic for selection of HEK-HiBiT-β₂AR positive cell clones (150 μg/ml zeocin).

To measure membrane localization of WT-CFTR-HiBiT protein in cells, Nano-Glo HiBiT Extracellular Detection System (Promega) was used according to the manufacturer’s protocol with a small alteration. Cells were seeded and incubated the same way as mentioned above in the lytic assay protocol. The medium was removed before adding a mixed reagent containing an assay buffer with LgBiT, substrate, and fresh medium without FBS. We optimized the total assay volume for both lytic and extracellular assays to 100 μl for 96-well plates and 25 μl for 384-well plates. The luminescent signal was measured by EnVision plate reader (Perkin Elmer).