Pbifc cc155

Manufactured by Addgene

PBiFC-CC155 is a plasmid designed for bimolecular fluorescence complementation (BiFC) analysis. It contains the C-terminal fragment of the fluorescent protein Venus, which can be used to detect protein-protein interactions when fused to a protein of interest.

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Lab products found in correlation

Pbifc vn173, by Addgene (2 mentions) Gateway lr clonase 2, by Thermo Fisher Scientific (1 mentions) Gateway cloning system, by Thermo Fisher Scientific (1 mentions)

3 protocols using pbifc cc155

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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Protein-Protein Interaction Verification via BiFC

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A BiFC assay was conducted to verify that TRAF6 and IRAK1 interacted with each other. TRAF6 and IRAK1 cDNA were inserted into the vectors pBiFC-VN173 (Addgene #22010) and pBiFC-CC155 (Addgene #22015) to construct the plasmids. HEK293T cells were transfected for 24 h with pBiFC-VN173-TRAF6, pBiFC-VN173-IRAK1, pBiFC-CC155-TRAF6, and pBiFC-CC155-IRAK1 alone; cotransfected with pBiFC-VN173-TRAF6 and pBiFC-CC155-IRAK1; or cotransfected with pBiFC-VN173-IRAK1 and pBiFC-CC155-TRAF6. The fluorescence intensity was then determined with a laser-scanning fluorescence microscope.

Xu C., Wang P., Guo H., Shao C., Liao B., Gong S., Zhou Y., Yang B., Jiang H., Zhang G, & Wu N. (2023). MiR-146a-5p deficiency in extracellular vesicles of glioma-associated macrophages promotes epithelial-mesenchymal transition through the NF-κB signaling pathway. Cell Death Discovery, 9, 206.

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