Materials and Reagents

1.5 mL tubes (WATSON, catalog number: 8064131815C)
0.22 μm filter (AS ONE, catalog number: 033022SO-SFCA)
6 cm cell culture dish (Thermo Scientific, catalog number: 150462)
Cell scraper (VIOLAMO, catalog number: 1-2249-01)
White 384-well immuno plates (Thermo Scientific, catalog number: 460372)
293T cells (ATCC: CRL-3216)
Plasmid pCAG-NS4B-FLAG-HiBiT, which encodes HiBiT-tagged JEV NS4B protein (Arakawa et al., 2022)
Plasmid pCAG-OSF-VCP, which encodes One Strep–tagged VCP proteins or is empty (Arakawa et al., 2022)
siRNA-luciferase, which targets firefly luciferase (sense sequence: 3′-CGUACGCGGAAUACUUCGAtt-5′)
siRNA-NPL4, which targets NPL4 (sense sequence: 3′-CUGAAGUGGUCGAUGAAAUtt-5’)
Nano Glo HiBiT lytic detection system (Promega, catalog number: N3040)
Dulbecco’s modified Eagle’s medium (Nacalai Tesque Inc., catalog number: 08458-16)
Fetal bovine serum (FBS) (Thermo Scientific, catalog number: 10270-106)
Phosphate-buffered saline (PBS) without calcium and magnesium (Nacalai Tesque Inc., catalog number: 14249-24)
Benzylpenicillin potassium (Fujifilm Wako Pure Chemical Corporation, catalog number: 021-07732)
Streptomycin sulfate (Tokyo Chemical Industry, catalog number: S0585)
Strep-Tactin Sepharose 50% suspension (IBA Lifesciences GmbH, catalog number: 2-121-010)
Strep-tag elution (10× buffer E) (IBA Lifesciences GmbH, catalog number: 2-1000-025)
cOmplete, EDTA-free, protease inhibitor cocktail (Roche, catalog number: 11873580001)
Lipofectamine^TM 3000 transfection reagent (Thermo Scientific, catalog number: L3000015)
Lipofectamine^TM RNAiMAX transfection reagent (Thermo Scientific, catalog number: 13778150)
Opti-MEM (Thermo Scientific, catalog number: 31985062)
Tris (Tris[hydroxymethyl]aminomethane) (Nacalai Tesque Inc., catalog number:35406-91)
NaCl (Nacalai Tesque Inc., catalog number: 31320-05)
Triton X-100 (Nacalai Tesque Inc., catalog number: 35501-15)
100× penicillin G + streptomycin stock solution (see Recipes)
Culture medium (see Recipes)
Pull-down washing buffer (see Recipes)
100× concentrated cOmplete stock solution (see Recipes)
Lysis buffer (see Recipes)
HiBiT reagent (see Recipes)
1× Strep-tag elution buffer (see Recipes)

Equipment

Humidified incubator (37 °C, 5% CO₂)
Microplate luminometer (Thermo Scientific, Varioskan LUX Multimode Microplate Reader)
Vortex mixer (Scientific Industries, model: Vortex-Genie 2)
Centrifuge machine for microtube (Thermo Scientific, model: Sorvall^TM Legend^TM Micro 21R)
Rotator (BIO CRAFT, model: BC-710I)

Procedure

Add 60 pmol siRNA-NPL4 (or siRNA-luciferase for negative control) to 250 µL of Opti-MEM. Mix well using a vortex mixer (Figure 1).
Note: Steps 1–8 pertain specifically to the siRNA transfection protocol, while step 9 marks the beginning of the pull-down protocol. To perform siRNA transfection, please follow the "Reverse transfection protocol" of Lipofectamine^TM RNAiMAX transfection reagent.

Figure 1. Overview of the protein pull-down HiBiT assay. A. Schematic illustration of the protein pull-down HiBiT assay. B. Flow diagram of the protein pull-down HiBiT assay.
Add 6 µL of Lipofectamine^TM RNAiMAX transfection reagent to 250 µL of Opti-MEM. Mix well using a vortex mixer.
Add the mixture from step 1 to that from step 2 and mix well using a vortex mixer.
Incubate the mixture for 5 min at room temperature.
Seed 293T cells in a 6 cm dish at a density of 750,000 cells/well in 4.5 mL of culture medium.
Gently add approximately 500 µL of the siRNA-Lipofectamine mixture to the cell-containing dish.
Place the dish in a CO₂ incubator for 24 h.
Replenish culture medium at 24 h post transfection.
Note: Slowly add medium to the side of the well to avoid cell detachment.
Add 1.5 µg pCAG-NS4B-FLAG-HiBiT, 1.5 µg pCAG-OSF-VCP, and 120 pmol siRNA-NPL4 (or siRNA-luciferase for negative control) to 250 µL of Opti-MEM. For siRNA transfection, repeat the initial transfection using the same siRNA. Mix well using a vortex mixer.
(Optional) Set control experiments using an empty vector (pCAG-OSF) in place of pCAG-OSF-VCP.
Add 6 µL of Lipofectamine^TM 3000 transfection reagent to 250 µL of Opti-MEM. Mix well using a vortex mixer.
Add the mixture from step 9 to that from step 10 and mix well using a vortex mixer.
Incubate the mixture for 10 min at room temperature.
Apply the mixture from step 12 to the cell-containing dish (~500 µL).
Place the dish in a CO₂ incubator for 48 h.
Aspirate the culture medium and add 1 mL of ice-cold PBS to each dish. Collect the cells using a cell scraper and transfer them to a 1.5 mL tube.
Centrifuge the 1.5 mL tubes at 500 × g for 10 min at 4 °C.
Aspirate PBS. Add 1 mL of ice-cold PBS to each tube and suspend by pipetting.
Centrifuge the 1.5 mL tubes at 500 × g for 10 min at 4 °C.
Aspirate PBS. Add 500 µL of ice-cold lysis buffer to each tube and mix by pipetting.
Centrifuge the 1.5 mL tubes at 20,000 × g for 10 min at 4 °C.
Transfer 5 µL of the supernatant to 1.5 mL tubes for input and add 45 µL of lysis buffer. Mix well using a vortex mixer.
Transfer 10 μL of diluted lysate in each well of a 384-well plate.
Note: To ensure accurate measurements, it is recommended to use more than three wells per sample. If the obtained values are outside the reading range, a dilution series should be carried out.
Add 10 μL of HiBiT reagent to each well.
Incubate for 10 min at room temperature.
Measure the luminescence for 1,000 ms measurement time/well using a microplate reader.
Transfer 480 µL of the clear lysate to fresh 1.5 mL tubes.
Add 15 µL of Strep-Tactin Sepharose 50% suspension to the lysate and rotate at 4 °C for 1 h.
Centrifuge the 1.5 mL tubes at 20,000 × g for 1 min at 4 °C.
Aspirate the supernatant. Add 1 mL of cold pull-down washing buffer to each tube and mix via inversion.
Repeat steps 28 and 29 thrice.
Centrifuge the 1.5 mL tubes at 20,000 × g for 1 min at 4 °C.
Aspirate the supernatant. Add 450 µL of 1× Strep-tag elution and rotate at 4 °C for 1 h.
Centrifuge the 1.5 mL tubes at 20,000 × g for 1 min at 4 °C.
Transfer 400 µL of elution buffer to fresh 1.5 mL tubes.
Transfer 10 μL of elution in each well of a 384-well plate.
Note: To ensure accurate measurements, it is recommended to use more than three wells per sample. If the obtained values are outside the reading range, a dilution series should be carried out.
Add 10 μL of HiBiT reagent from Nano Glo HiBiT lytic detection system to each well.
Incubate for 10 min at room temperature.
Measure the luminescence for 1,000 ms measurement time/well using a microplate reader (Figure 2).

Figure 2. HiBiT activity of the Strep-Tactin-purified fraction and the input fraction. A. HiBiT activity of the Strep-Tactin-purified fraction. B. HiBiT activity of the input fraction. C. The compensated HiBiT value of the Strep-Tactin purified fraction. The HiBiT activity of the Strep-Tactin purified fraction was calculated by dividing its value by that of the input fraction. The means between two groups were compared using a Student’s t-test. Differences were considered significant at **P < 0.01.

Notes

In the NanoLuc/HiBiT system, strong luminescence emission may be obtained and causes leaks to adjacent wells in a multi-well plate. Therefore, we recommend loading the samples in every other well.
The materials used in this study can be obtained from the corresponding author, Eiji Morita, upon reasonable request.

Recipes

100× penicillin G + streptomycin stock solution
PBS 100 mL
Benzylpenicillin potassium 0.626 g
Streptomycin sulfate 1 g
Sterilize using a 0.22 μm filter and store in a refrigerator.

Culture medium
Dulbecco’s modified Eagle’s medium 500 mL
FBS, heat-inactivated via incubation at 56 °C for 45 min 50 mL
100× penicillin G + streptomycin stock solution 5 mL

Pull-down washing buffer
1 M Tris pH 7.5 20 mL
5 M NaCl 30 mL
Triton X-100 1.07 g
ddH₂O up to 1 L

100×-concentrated cOmplete stock solution
cOmplete, EDTA-free, protease inhibitor cocktail 1 tablet
ddH₂O 500 µL
Store at -20

Lysis buffer
1 M Tris pH 7.5 20 mL
5 M NaCl 30 mL
Triton X-100 10.7 g
ddH₂O up to 1 L
Add 100× concentrated cOmplete stock solution just before use.

HiBiT reagent from Nano Glo HiBiT lytic detection system
Nano-Glo HiBiT lytic buffer 500 µl
Nano-Glo HiBiT lytic substrate 10 µl
LgBiT protein 5 µl

1× Strep-tag elution buffer
Strep-tag elution (10× buffer E) 2 mL
ddH₂O 18 mL

Acknowledgments

This work was supported by JSPS KAKENHI (Grant Number 22H00553, 22H02873, 22K18378, 20H05305, 20K21874), JST CREST, Japan (grant number JPMJCR17H4). The protocol for HiBiT detection was adapted from previous work (Goto et al., 2020). This protocol is derived from the original research paper (Arakawa et al., 2022; DOI: 10.1016/j.jbc.2022.101597).

Competing interests

The authors have no competing interests directly relevant to the content of this article.

References

Arakawa, M. and Morita, E. (2019). Flavivirus Replication Organelle Biogenesis in the Endoplasmic Reticulum: Comparison with Other Single-Stranded Positive-Sense RNA Viruses. Int J Mol Sci 20(9).
Arakawa, M., Tabata, K., Ishida, K., Kobayashi, M., Arai, A., Ishikawa, T., Suzuki, R., Takeuchi, H., Tripathi, L. P., Mizuguchi, K., et al. (2022). Flavivirus recruits the valosin-containing protein-NPL4 complex to induce stress granule disassembly for efficient viral genome replication. J Biol Chem 298(3): 101597.
Dixon, A. S., Schwinn, M. K., Hall, M. P., Zimmerman, K., Otto, P., Lubben, T. H., Butler, B. L., Binkowski, B. F., Machleidt, T., Kirkland, T. A., et al. (2016). NanoLuc Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells. ACS Chem Biol 11(2): 400-408.
Tabata, K., Arakawa, M., Ishida, K., Kobayashi, M., Nara, A., Sugimoto, T., Okada, T., Mori, K. and Morita, E. (2021). Endoplasmic Reticulum-Associated Degradation Controls Virus Protein Homeostasis, Which Is Required for Flavivirus Propagation. J Virol 95(15): e0223420.
Goto, S., Ishida, K., Suzuki, R. and Morita, E. (2020). Split Nano Luciferase-based Assay to Measure Assembly of Japanese Encephalitis Virus. Bio Protoc 10(9): e3606.