Pbifc vn173

Manufactured by Addgene

Sourced in Japan, United States

PBiFC-VN173 is a lab equipment product. It is a vector that expresses the VN173 fragment of the Venus fluorescent protein, which can be used for bimolecular fluorescence complementation (BiFC) assays to study protein-protein interactions.

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Lab products found in correlation

Pbifc vc155, by Addgene (15 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (4 mentions) Lipofectamine 3000, by Thermo Fisher Scientific (4 mentions) Pbifc cc155, by Addgene (2 mentions) Gateway lr clonase 2, by Thermo Fisher Scientific (1 mentions) Gateway cloning system, by Thermo Fisher Scientific (1 mentions) Confocal microscope, by Nikon (1 mentions) Ha ubiquitin, by Addgene (1 mentions) Ha ubiquitin k48r, by Addgene (1 mentions) Ha ubiquitin k48, by Addgene (1 mentions) Rnaimax, by Thermo Fisher Scientific (1 mentions) Turbofect transfection reagent, by Thermo Fisher Scientific (1 mentions) Neuroporter transfection kit, by Merck Group (1 mentions) Quikchange multi site directed mutagenesis kit, by Agilent Technologies (1 mentions) Pmcherry n1 plasmid, by Takara Bio (1 mentions) Facsaria 2, by BD (1 mentions) Genetailor site directed mutagenesis system, by Thermo Fisher Scientific (1 mentions) A1si laser scanning confocal microscope, by Nikon (1 mentions) A1rsi confocal microscope, by Nikon (1 mentions)

18 protocols using pbifc vn173

Constructing SARS-CoV-2 Spike Expression Plasmids

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pCMV3-SARS-CoV-2-spike plasmid (pCMV3-Spike) is a gift from Ron Diskin lab. Via PCR we truncated 19AA from C-terminal part or added Flag-tag at C-terminal part and subcloned it back in the same plasmid backbone (pCMV3-Δ19Spike or pCMV3-SARS-Spike-Flag). For the construction of pLenti6-hACE2-4xmyc we first cloned hACE2-4xmyc first into pENTR plasmid with restriction sites NcoI and KpnI. Afterwards we used restriction-free cloning method via Gateway^® LR-clonase-II (Invitrogen) and the manufactural protocol.
For pEFIRES-hACE2-4xMyc we used restriction sites NheI and MluI and for pEFIRES-TMPRSS2-Flag NheI and XbaI restriction sites. For cell-fusion-assay, pBiFC-VN173 (Addgene) was used to fuse Jun upstream to YFPn (Jun-YFPn), using HindIII and BglII restriction sites. pBiFC-CC155 (Addgene) was used to fuse Fos upstream to the YFPc (Fos-YFPc) using EcoRI and KpnI restriction sites.

Strobelt R., Adler J., Paran N., Yahalom-Ronen Y., Melamed S., Politi B., Shulman Z., Schmiedel D, & Shaul Y. (2022). Imatinib inhibits SARS-CoV-2 infection by an off-target-mechanism. Scientific Reports, 12, 5758.

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Protein-protein Interaction Assay with Split Luciferase

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PSMA subunits (kindly provided by Prof. K. Tanaka, Tokyo Metropolitan Institute of Medical Science, Japan) were cloned into pBiFC-VN173 (Addgene plasmid no. 22010), a gift from Prof. Chang-Deng Hu (Purdue University). The 6xmyc p21 was cloned into pBiFC-CC155 (Addgene plasmid no. 22015), a gift from Prof. Chang-Deng Hu. Luc1 and Luc2, the respective N’ terminal and C’ terminal fragments of split Gaussia luciferase, were kindly provided by Prof. Adi Kimchi (Weizmann Institute of Science, Israel.). HEK293 cells were transfected using the calcium phosphate method [26 (link)]. U2OS cells stably expressing the chimeric PSMA3 subunit were created using the Gateway cloning system (Invitrogen, Carlsbad, CA, USA).

Biran A., Myers N., Steinberger S., Adler J., Riutin M., Broennimann K., Reuven N, & Shaul Y. (2022). The C-Terminus of the PSMA3 Proteasome Subunit Preferentially Traps Intrinsically Disordered Proteins for Degradation. Cells, 11(20), 3231.

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Bimolecular Fluorescence Complementation Assay

Cited 3 times

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Human p113 ORF (342 bp), ZRF1 cDNA (1866 bp), and BRD4 cDNA (4089 bp) were subcloned into pBiFC-VN173 or pBiFC-VC155 (Addgene), and co-transfected into tumor cells with Lipofectamine 2000 (Invitrogen) for 24 h. The fluorescence was observed with a confocal microscope (Nikon, Japan) [15 (link), 16 (link), 19 (link)].

Yang F., Hu A., Guo Y., Wang J., Li D., Wang X., Jin S., Yuan B., Cai S., Zhou Y., Li Q., Chen G., Gao H., Zheng L, & Tong Q. (2021). p113 isoform encoded by CUX1 circular RNA drives tumor progression via facilitating ZRF1/BRD4 transactivation. Molecular Cancer, 20, 123.

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Plasmid-based Ubiquitin Interaction Analysis

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Plasmid encoding HA‐Ubiquitin, HA‐Ubiquitin‐K48, HA‐Ubiquitin‐K48R were purchased from Addgene and used directly without modification. Plasmid encoding pBiFC‐VC155, pBiFC‐VN173, MDM2‐YFP were purchased from Addgene and used as templates for subcloning. The plasmids were constructed for this study as described in “KEY RESOURCES TABLE” (Supporting Information). All plasmid inserts were validated by sequencing at Genewiz. All siRNAs were purchased from Synbio Technologies. The sequences of the siRNA were described in “KEY RESOURCES TABLE” (Supporting Information). Cultured cells were transfected with different plasmids using Lipofectamine 2000 (Invitrogen, 11668‐019) and with siRNAs using RNAi Max (Invitrogen, 13778150) according to the manufacturer's instructions.

Li Y., Fu Y., Zhang Y., Duan B., Zhao Y., Shang M., Cheng Y., Zhang K., Yu Q, & Wang T. (2023). Nuclear Fructose‐1,6‐Bisphosphate Inhibits Tumor Growth and Sensitizes Chemotherapy by Targeting HMGB1. Advanced Science, 10(7), 2203528.

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Evaluating Protein-Protein Interactions

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Primers for p57, p53, Pcna, p21, p27, p16, Wnt6, and Wnt2 were designed using primer 5 and are listed in Table S2. For BiFC assay, p57 gene was linked with pBiFC-VC155 (Addgene, Watertown, Massachusetts, USA), while p53, Pcna, p21, p27, p16, Wnt6, and Wnt2 genes were linked with pBiFC-VN173 (Addgene) as described above. Before transfection, 1 × 10⁵ 293 T cells were plated into each well of 12-well plates. BiFC-p57-VC155 was cotransfected with BiFC-p53-VN173 (or BiFC-Pcna-VN173, BiFC-p21-VN173, BiFC-p27-VN173, BiFC-p16-VN173, BiFC-Wnt6-VN173, and BiFC-Wnt2-VN173) using TurboFect Transfection Reagent (Thermo Fisher Scientific) according the manufacturer's instructions. 6 h later, fresh culture medium was changed and the cells were further cultured for 48 h before analysis.

Li N., Du Z., Li Y., Xu W., Yang Y., Peng H., Song T., Qin Q., Lei H, & Hua J. (2021). p57 Suppresses the Pluripotency and Proliferation of Mouse Embryonic Stem Cells by Positively Regulating p53 Activation. Stem Cells International, 2021, 4968649.

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Bimolecular Fluorescence Complementation of Oncogenes

Cited 2 times

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Human cDNAs of c‐Myc (1365 bp), LMNA (1995 bp) and LMNC (1719 bp) were inserted into pBiFC‐VN173 (containing Flag‐tag) or pBiFC‐VC155 (containing HA‐tag) from Addgene. The c‐Myc and LMNA constructs were mutated by QuikChange Multi Site‐Directed Mutagenesis Kit (Agilent) using primer sets (Table S5). Constructs were co‐transfected into tumour cells by using NeuroPorter Transfection Kit (Sigma) for a 24‐h period, while fluorescence imaging was detected using a confocal microscope.
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Wang J., Hong M., Cheng Y., Wang X., Li D., Chen G., Bao B., Song J., Du X., Yang C., Zheng L, & Tong Q. (2024). Targeting c‐Myc transactivation by LMNA inhibits tRNA processing essential for malate‐aspartate shuttle and tumour progression. Clinical and Translational Medicine, 14(5), e1680.

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BiFC Assay for DDI2ΔUBL Interaction

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The BiFC assay was performed as described previously.⁶⁷ (link) To generate plasmids expressing split Venus fragments-tagged DDI2ΔUBL, the DDI2 cDNA was cloned into pBiFC-VN173 (Addgene_22010) or pBiFC-VC155 (Addgene_22011), respectively. Both BiFC plasmids were co-transfected with a pmCherry-N1 plasmid (Clontech), which was used as an internal control. At one day after transfection, the cells were cultured without all amino acids for 3 h. The fluorescent intensities of Venus and mCherry were measured using a flow cytometer (FACSAriaII, BD Biosciences). Median fluorescence intensity (MFI) values of Venus in mCherry-positive cells were measured by flow cytometry.

Hirose S., Waku T., Tani M., Masuda H., Endo K., Ashitani S., Aketa I., Kitano H., Nakada S., Wada A., Hatanaka A., Osawa T., Soga T, & Kobayashi A. (2023). NRF3 activates mTORC1 arginine-dependently for cancer cell viability. iScience, 26(2), 106045.

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Fluorescent Protein Interaction Assay

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Human EWSR1 cDNA (1,971 bp) and MAZ cDNA (1,482 bp) were subcloned into BiFC vectors pBiFC‐VN173 and pBiFC‐VC155 (Addgene), and co‐transfected into tumor cells for 24 h. The fluorescence emission was observed under a confocal microscope, with excitation and emission wavelengths of 488 and 500 nm, respectively (Kerppola, 2008).

Li H., Yang F., Hu A., Wang X., Fang E., Chen Y., Li D., Song H., Wang J., Guo Y., Liu Y., Li H., Huang K., Zheng L, & Tong Q. (2019). Therapeutic targeting of circ‐CUX1/EWSR1/MAZ axis inhibits glycolysis and neuroblastoma progression. EMBO Molecular Medicine, 11(12), e10835.

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Bimolecular Fluorescence Complementation

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Human ARMC12 cDNA (1104 bp) or RBBP4 cDNA (1278 bp) was respectively subcloned into pBiFC-VN173 and pBiFC-VC155 (Addgene, Cambridge, MA). Mutation of ARMC12 or RBBP4 was undertaken with GeneTailor^TM Site-Directed Mutagenesis System (Invitrogen) and PCR primers (Supplementary Table 7). After co-transfection of recombinant constructs using Lipofectamine 3000 (Invitrogen) for 24 h, tumor cells were grown for additional 10 h at 37 °C. Under a confocal microscope, the fluorescence emission was detected (488 and 500 nm as excitation and emission wavelengths, respectively).

Li D., Song H., Mei H., Fang E., Wang X., Yang F., Li H., Chen Y., Huang K., Zheng L, & Tong Q. (2018). Armadillo repeat containing 12 promotes neuroblastoma progression through interaction with retinoblastoma binding protein 4. Nature Communications, 9, 2829.

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Visualizing hnRNPU and CTCF Interaction

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Human hnRNPU cDNA (2478 bp) and CTCF cDNA (2184 bp) were subcloned into bimolecular fluorescence complementation (BiFC) vectors pBiFC-VN173 and pBiFC-VC155 (Addgene) and co-transfected into tumor cells with Lipofectamine 2000 (Invitrogen) for 24 h. Fluorescence emission was observed under a confocal microscope, using excitation and emission wavelengths of 488 and 500 nm, respectively [14 (link)].

Song H., Li D., Wang X., Fang E., Yang F., Hu A., Wang J., Guo Y., Liu Y., Li H., Chen Y., Huang K., Zheng L, & Tong Q. (2020). RETRACTED ARTICLE: HNF4A-AS1/hnRNPU/CTCF axis as a therapeutic target for aerobic glycolysis and neuroblastoma progression. Journal of Hematology & Oncology, 13, 24.

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