All eIF4E, eIF4G (including the Y624A, L629A and L630A binding mutant) and 4EBP1 mutant cDNAs were synthetized and obtained from IDT (Integrated DNA Technologies). eIF4E and eIF4G604–646 were cloned into NanoBit plasmids using the NanoBit PPI starter system (Promega) using XhoI/EcoRI and NheI/EcoRI cloning sites respectively. 4EBP1 mutants were cloned into pCDNA3.1 vector DNA (Thermo Fisher Scientific) harbouring a C-terminal 3× FLAG tag via NheI/BamHI sites to allow mammalian cell overexpression. For bacterial expression, 4EBP1 mutants were cloned into pGEX6P1 using BamHI/EagI cloning sites. GFP and v-Myc coding sequence residing in a pCMV6 mammalian expression vector were obtained from Origene. The bicistronic luciferase reporter construct pcDNA3-rLuc-polIRES-fLuc was purchased from Addgene. PP242, Torin1, Rapamycin, 4EGi-1 and 4E1RCat were purchased from Tocris Bioscience, whilst all other chemicals unless otherwise stated were purchased from Selleck Chemicals. siRNAs targeting either 4EBP1 (ON-TargetPlus Human EI4EBP1, J-003005-12-0005) or non-targeting control (ON-TargetPlus Non-targeting pool, D-001810-10-05) were purchased from DHARMACON.
Nanobit ppi starter system
Manufactured by Promega
Sourced in United States, Japan
The NanoBit PPI Starter System is a tool designed for the detection and analysis of protein-protein interactions (PPIs). It utilizes NanoBit technology, which involves the splitting of a luminescent protein reporter into two complementary fragments. When these fragments are brought together by interacting proteins, the luminescent signal is restored, allowing for the visualization and quantification of PPIs.
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Lab products found in correlation
Rapamycin, by Bio-Techne (1 mentions)
Torin1, by Bio-Techne (1 mentions)
Pcmv6 mammalian expression vector, by OriGene (1 mentions)
Pcdna3 rluc polires fluc, by Addgene (1 mentions)
4e1rcat, by Bio-Techne (1 mentions)
On targetplus non targeting pool d 001810 10 05, by Horizon Discovery (1 mentions)
4egi 1, by Bio-Techne (1 mentions)
Pp242, by Bio-Techne (1 mentions)
Cxcl12, by Thermo Fisher Scientific (1 mentions)
Nano glo live cell assay system, by Promega (1 mentions)
One glo luciferase assay system, by Promega (1 mentions)
Pgl4.33 luc2p sre hygro, by Promega (1 mentions)
Pbit3.1 plasmid, by Promega (1 mentions)
Nano glo hibit extracellular detection system, by Promega (1 mentions)
Restriction enzyme, by New England Biolabs (1 mentions)
Anti ha antibody, by Merck Group (1 mentions)
Restriction enzyme, by Takara Bio (1 mentions)
Pnl1.1 nluc vector, by Promega (1 mentions)
Fluo 4 acetoxymethyl ester, by Thermo Fisher Scientific (1 mentions)
Nano glo luciferase assay system, by Promega (1 mentions)
8 protocols using nanobit ppi starter system
1
Characterizing eIF4E-eIF4G and 4EBP1 Interactions
2
Cloning and Engineering of Sterol Biosynthesis Genes
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The protein-coding sequences of NSDHL, SC4MOL, and HSD17B7 were amplified from HeLaT cDNA and cloned into our in-house pcDNA5/FRT construct (33 ) with a C-terminal V5 tag, with a CMV promoter. NSDHL was then moved to a construct with a weaker TK promoter to reduce expression levels. The pcDNA3.1-DHCR24-V5, pcDNA3.1-V5-DHCR24 (34 ), and pcDNA3.1-SM-V5 (7 (link)) were previously generated. NanoBiT® plasmids were cloned following the protocol outlined in the NanoBiT® PPI Starter System (Promega). SC4MOL NanoBiT® constructs were previously generated (15 (link)). SM-V5 (7 (link)) was amplified by PCR and subcloned using SacI and NheI restriction enzyme cloning into plasmids supplied in the kit [pBiT1.1-C (TK/LgBiT), pBiT2.1-C (TK/SmBiT), pBiT1.1-N (TK/LgBiT), or pBiT2.1-N (TK/SmBiT)]. MARCHF6-V5 (35 (link)) constructs were made similarly, but with NheI and EcoRI restriction enzymes. All plasmids were confirmed via Sanger sequencing conducted by the Ramaciotti Centre for Genomics at UNSW Sydney. Primer sequences are provided in supplemental Table S1 .
3
Bimolecular Fluorescence Complementation Assay for PKA
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NanoBit PPI Starter System (N2014, Promega) was used to generate GlPKA-NBit. Briefly, the complimentary peptide Small BiT (SmBiT; 11 amino peptide) was fused with PKAc (GL50803_101214) driven by its native promoter (~500 bp), and the Large BiT (LgBiT; 18kDa) was fused to PKAr (GL50803_9117) driven by its native promoter (~500 bp) (Fig. 2c , Supplementary Fig. 2a ). The control cell line expresses the PKAc-SmBiT and the LgBiT fragment driven by the PKAr native promoter (pPKAr) not fused to any protein (Supplementary Fig. 2a ).
4
Detecting HBV Protein Interactions using NanoBiT
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The NanoBiT PPI Starter System (catalog #N2014; Promega) was used for the detection of interaction between HBc and L-HBs proteins. To generate the HBc-LgBiT plasmid, the HBc region of HBV genotype C (accession number AB246344 ) was amplified by PCR with appropriate primers and inserted into pBiT1.1-C after digestion with EcoRI and XhoI (Figure 5 A). To generate the SmBiT-L-HBs plasmid, the L-HBs region of the same HBV clone was amplified by PCR with appropriate primers and inserted into pBiT2.1-N after digestion with XhoI and EcoRI. The HBV 1.4-fold [ΔHBe/c, ΔpreS1] plasmid was also generated by modifying the first methionine of ORFs of HBe, HBc, and preS1 regions to erase the expression of HBe, HBc, and L-HBs proteins of the replication-competent HBV genotype C molecular clone. The addition of this plasmid to the plasmids for the expression of HBc and L-HBs proteins enables the enhancement of the interaction signal by mimicking the assembly of viral particles. The intracellular NanoLuc signal generated by the interaction between LgBiT and SmBiT was quantified 3 days after transfection.
5
CXCL12-mediated CXCR4 signaling assays
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CXCL12 was purchased from Peprotech (Rocky Hill, NJ, USA). NanoBiT® PPI starter system and Nano-Glo® live cell assay system; pBiT3.1 plasmid and Nano-Glo® HiBiT extracellular detection system; and pGL4.33 [luc2P SRE Hygro] and ONE-Glo luciferase assay system were purchased from Promega (Madison, WI, USA). Restriction enzymes were obtained from New England Bio Labs (Ipswich, MA, USA). Anti-CXCR4 antibody (Cat. No. ab124824) was purchased from Abcam (Toronto, ON, Canada). Anti-ERK antibody (Cat. No. 4695) and anti-pERK antibody (Thr202/Tyr204) (Cat. No. 4370) were obtained from Cell Signaling Technology (Beverly, MA, USA). Anti-HA antibody (Cat. No. H3663) and anti-FLAG M2 affinity gel (Cat. No. A2220) for co-IP were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-Green fluorescence protein (GFP) antibody (Cat. No. sc-8334), anti-β-actin antibody (Cat. No. sc-9996), and all secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All chemicals were obtained from Sigma-Aldrich unless otherwise stated.
6
PKA Protein-Protein Interaction Assay
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NanoBit PPI Starter System (N2014, Promega) was used to generate GlPKA-NBit. Briefly, the complimentary peptide Small BiT (SmBiT; 11 amino peptide) was fused with PKAc driven by its native promoter (~500 bp), and the Large BiT (LgBiT; 18kDa) was fused to PKAr driven by its native promoter (~500 bp) (Fig. 2c , Supplementary Fig. 2a ). The control cell line expresses the PKAc-SmBiT and the LgBiT fragment driven by the PKAr native promoter (pPKAr) not fused to any protein (Supplementary Fig. 2a ). Primer sequences are in Supplementary File1 .
7
Fluorescent Protein Gene Expression Protocol
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The Venus fluorescent protein gene was provided by Dr. A. Miyawaki (RIKEN, Japan)48 (link). Morphine hydrochloride was purchased from Takeda Pharmaceutical. [Arg8]-Vasopressin was purchased from Peptide Institute, Inc. (Osaka, Japan). DAMGO was purchased from Sigma-Aldrich (St. Louis, MO, USA). Fluo-4 acetoxymethyl ester was purchased from Thermo Fisher Scientific (Tokyo, Japan). The FuGENE HD, NanoGlo Luciferase Assay System, pNL1.1[Nluc] vector, and NanoBit PPI Starter System were purchased from Promega (Tokyo, Japan). The Plasmid Midi Kit was obtained from QIAGEN. Escherichia coli DH5α competent cells were obtained from Takara Bio (Kusatsu, Japan). Restriction enzymes were obtained from Takara Bio and New England Biolabs Japan, Inc. (Tokyo, Japan). The anti-HA antibody was purchased from Sigma-Aldrich. All other chemicals were of reagent grade (Wako Pure Chemical Industries, Osaka, Japan).
8
Subcloning of JAK and Receptor Proteins
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JAK1, JAK2, JAK3, and TYK2 were subcloned in the pMX-internal ribosomal entry site (IRES)-green fluorescent protein (GFP) and EpoR, EpoR:gp130, IFN-a/b receptor 1/2, IFNGR1/2, IL-2R, and IL-9R in the pMX-IRES-CD4. 27 The JAK2 JH2 domain (amino acids 535-812) was subcloned in pFast-Bac1 vector (Life Technologies, Grand Island, NY). Sitedirected mutagenesis was performed, as previously described. 11 Jak2 cDNA was cloned into pHT-C and pNL-C vectors from the NanoBRET PPI starter system (N1821) from Promega (Leiden, The Netherlands) by using NheI and SacI restriction sites. EpoR was cloned into both pBiT-C-LgBiT 1.1 and pBiT-C-SmBiT 2.1 vectors from the NanoBiT PPI starter system (N2014; Promega, Fitchburg, Wis) by using NheI and EcoRI restriction sites. Sequences were verified by means of Sanger sequencing.
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