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Prk5 vector

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The PRK5 vector is a plasmid used for gene expression in mammalian cells. It contains a CMV promoter and a multiple cloning site for the insertion of target genes. The vector also includes a neomycin resistance gene for selection of transfected cells.

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Lab products found in correlation

Anti myc antibody, by Cell Signaling Technology (2 mentions) Anti flag beads, by Merck Group (2 mentions) Anti flag, by Merck Group (2 mentions) A2220, by Merck Group (1 mentions) Xp04205box, by Thermo Fisher Scientific (1 mentions) F7425, by Merck Group (1 mentions) Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions) Protease inhibitor, by Merck Group (1 mentions) Fugene hd, by Promega (1 mentions) Quikchange lightning site directed mutagenesis kit, by Agilent Technologies (1 mentions) Nhei hf, by New England Biolabs (1 mentions) Pcdna3, by Thermo Fisher Scientific (1 mentions) Xbai, by New England Biolabs (1 mentions) Lipofectamine, by Thermo Fisher Scientific (1 mentions)

7 protocols using prk5 vector

1

Affinity Purification of FLAG-FAM46A/C and Myc-BCCIPα/β

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Full-length human FAM46A/FAM46C with a FLAG-tag and BCCIPα/BCCIPβ with a Myc-tag were cloned into the pRK5 vector (556104, BD PharMingen). The plasmids were transfected into HEK293T cells with Lipofectamine 3000 (L3000001, Invitrogen) according to the manufacturer’s instruction. Cells were harvested 36 hours after transfection and lysed in the lysis buffer containing 50 mM tris (pH 8.0), 150 mM NaCl, 0.1% NP-40, 1 mM DTT, and protease inhibitor (78442, Sigma-Aldrich). Lysates were cleared by centrifugation at 15,000 rpm for 10 min at 4°C. anti-FLAG beads (A2220, Sigma-Aldrich) were added to the supernatant, and the mixture was rotated at 4°C for 1 hour. The beads were washed with a lysis buffer three times. Proteins remaining on the beads were resolved with 4 to 20% gradient SDS-PAGE (XP04205BOX, Invitrogen) and detected by Western blot using anti-FLAG (F7425, Sigma-Aldrich) and anti-Myc antibodies (2278, Cell Signaling Technology).
Liu S., Chen H., Yin Y., Lu D., Gao G., Li J., Bai X.C, & Zhang X. (2023). Inhibition of FAM46/TENT5 activity by BCCIPα adopting a unique fold. Science Advances, 9(14), eadf5583.
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2

Immunoprecipitation of Myc-FAM46C and FLAG-Plk4

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Full-length human FAM46C with a Myc-tag and Plk4 with a FLAG-tag were cloned into the pRK5 vector (BD PharMingen, #556104). HEK293T cells were transfected with the plasmids with Fugene HD (Promega, #E2311) according to the manufacturer’s instruction. Cell were harvested 36 hours after transfection and lysed with a lysis buffer containing 50 mM Tris (pH 8.0), 150 mM NaCl, 0.1% NP-40, 1 mM DTT and protease inhibitor (Sigma, P8340). Lysates were centrifuged at 15000 rpm for 10 minutes at 4 °C, and the supernatant was transferred into a new tube. anti-FLAG beads (Sigma, #A2220) were added into the supernatant, and the mixture were rotated at 4 °C for 1 hour. The beads were washed with the lysis buffer three times. Proteins remaining on the beads were resolved with 4–20% gradient SDS-PAGE (Bio-Rad, #4561096) and detected by western blot using anti-FLAG (Sigma, #F1804) and anti-Myc antibodies (Cell Signaling Technology, #2276).
Chen H., Lu D., Shang G., Gao G, & Zhang X. (2020). Structural and functional analyses of the FAM46C/Plk4 complex. Structure (London, England : 1993), 28(8), 910-921.e4.
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3

LRP5 Plasmid Construction and Mutagenesis

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The gene encoding wild-type LRP5 (Origene, Rockville, MD) was subcloned in-frame into the pRK5 vector (BD Bioscience, San Jose, CA) using the XbaI and HindIII sites. FZD4 and Norrin cDNAs fused to reporter sequences/genes (generously provided by Dr. Jeremy Nathans) have been described previously [19 (link),30 (link)]. LRP5 mutations were introduced into the wild-type LRP5 cDNA by primer-mediated PCR mutagenesis using the QuikChange® Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, Inc., Santa Clara, CA). The recombinant plasmids containing LRP5-pRK-5 fusion constructs were verified by direct DNA sequencing and then amplified and purified for transfection (Tiangen, Biotech, Beijing, China).
Fei P., Zhang Q., Huang L., Xu Y., Zhu X., Tai Z., Gong B., Ma S., Yao Q., Li J., Zhao P, & Yang Z. (2014). Identification of two novel LRP5 mutations in families with familial exudative vitreoretinopathy. Molecular Vision, 20, 395-409.
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4

Construction of A1R and A2AR Fusion Proteins

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The cDNA encoding the human A1R tagged at its N-terminal tail with the O6-alkylguanine-DNA alkyltransferase (i.e., A1RSNAP) cloned in pRK5 vector (BD PharMingen, San Jose, CA, USA) was a gift from Prof. Jean-Philippe Pin (CNRS, Montpellier, France). Thus, to perform functional assays A2ARSNAP [24 (link)] and A1RSNAP were used. Also, A2ARRLuc and A1RYFP constructs [17 (link)] were used to perform classical BRET (Bioluminescence Resonance Energy Transfer) assays. Finally, to perform NanoBRET experiments with the MRS7396 fluorescent antagonist, we created an A2AR NanoLuc sensor (A2ARNL). To this end, the cDNA encoding the human A2AR was amplified by polymerase chain reaction from the pECFP-A2AR vector using the primers: FA2AEco (5′-GCCGGAATTCCCCATCATGGGCTCCTCGGTGTAC-3′) and RA2ANot (5′-CGCGGCGGCCGCtcaggacactcctgctccatcctggg-3′). The amplified A2AR insert was then cloned into the EcoRI/NotI sites of pNLF1-secN vector (Promega, Stockholm, Sweden) containing a hemagglutinin (HA) epitope tag. All the constructs were verified by DNA sequencing.
Lanznaster D., Massari C.M., Marková V., Šimková T., Duroux R., Jacobson K.A., Fernández-Dueñas V., Tasca C.I, & Ciruela F. (2019). Adenosine A1-A2A Receptor-Receptor Interaction: Contribution to Guanosine-Mediated Effects. Cells, 8(12), 1630.
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5

Cloning and expression of human GDF11 and FST

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The full coding sequence of human GDF11 was amplified by PCR using the following primers: 5’-CACCATGGTGCTCGCGGCCCCGCT-3’ and 5’-CGCTGTGGCTGCTCTTAA-3’. The product was then double-digested with NheI-HF and XbaI (New England Biolabs) and cloned into pcDNA3.1(+) (Life Technologies, Carlsbad, CA, USA). An alternate GDF11 expression construct was also made using the pRK5 vector (BD PharMingen) for experiments using HEK293T cells. Constructs for expression of wildtype human FST (isoform Fs288; in pcDNA3.1) (Cash et al., 2012a (link)) and human Furin (in pcDNA4; Walker et al., 2017 (link)) were previously described. Site-directed mutagenesis was employed to generate the respective expression vectors carrying the GDF11:c.893G>A and FST:c.167G>A variants. Separately, four SMAD-binding elements (SBE, 5’-CAGACA-3’) were synthesized (IDT, Coralville, IA, USA) and cloned into pENTR/D-TOPO and then shuttled into a cFos-FFLuc plasmid (SBE-FFLuc) for the in vitro luminescence reporter activity assay.
Cox T.C., Lidral A.C., McCoy J.C., Liu H., Cox L.L., Zhu Y., Anderson R.D., Uribe L.M., Anand D., Deng M., Richter C.T., Nidey N.L., Standley J.M., Blue E.E., Chong J.X., Smith J.D., Kirk E.P., Venselaar H., Krahn K.N., van Bokhoven H., Zhou H., Cornell R.A., Glass I.A., Bamshad M.J., Nickerson D.A., Murray J.C., Lachke S.A., Thompson T.B., Buckley M.F, & Roscioli T. (2019). Mutations in GDF11 and the extracellular antagonist, Follistatin, as a likely cause of Mendelian forms of orofacial clefting in humans. Human mutation, 40(10), 1813-1825.
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6

Characterization of mmCx36 Interactions

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Three different mmCx36 constructs (1. full-length mmCx36, 2. mmCx36 S315D, 3. mmCx36 S318 ter) were cloned into the pRK5 vector (BD Pharmingen, San Diego, CA, USA). The PDZ domain 10 of rnMUPP1 was cloned into the PetM11 vector. All constructs were sequenced for accuracy. PSD95-FLAG in pcDNA5/FRT/TO was a gift from Dr. Wei-dong Yao (Addgene plasmid #15463; RRID:Addgene_15463). This clone was initially generated by Zhang et al.37 (link). Monomeric eGFP-CaMKIIa fusion construct was a gift of Dr. M. Neal Waxham (University of Texas Health Science Center at Houston)38 (link). HEK293 and HeLa cells were plated at a density of 5 × 105 cells in a Petri dish (6 cm diameter), including 12 mm coverslips, in 5 ml Dulbecco’s Modified Eagle Medium. Cells were transfected with 0.6–1.4 µg/ml DNA using Lipofectamine (Life Technologies), 24 h after seeding. All transfections were done as triplicates and independently performed at least twice.
To stimulate endogenous CaMKII activity, transfected HeLa cells were treated with serum-free media containing 1 mM glycine and 100 µM glutamate for 15 min in a cell culture incubator (37 °C, 5% CO2). Afterwards, HeLa cells were fixed in 2% PFA in PBS for 15 min and prepared for confocal scans.
Tetenborg S., Wang H.Y., Nemitz L., Depping A., Espejo A.B., Aseervatham J., Bedford M.T., Janssen-Bienhold U., O’Brien J, & Dedek K. (2020). Phosphorylation of Connexin36 near the C-terminus switches binding affinities for PDZ-domain and 14–3–3 proteins in vitro. Scientific Reports, 10, 18378.
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7

Molecular Cloning of Tagged Proteins

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Total RNA was extracted from IPEC-J2 cells, and genomic cDNA was prepared using RT-PCR. FLAG-tagged Ythdf1 was cloned into the pCMV-Tag 4 vector (Agilent, 211174), and Myc-tagged Ddx60 was cloned into the pRK-5 vector (BD Pharmingen, 556104). We generated truncation plasmids based on the corresponding full-length cDNAs. All other plasmids, including full-length, coding sequence, and mutation plasmids, were generated using GenScript and cloned into the FLAG-pcDNA3.1 vector. All plasmids were confirmed by DNA sequencing. The primers used for cloning are listed in Supplementary Table S3.
Zong X., Xiao X., Shen B., Jiang Q., Wang H., Lu Z., Wang F., Jin M., Min J., Wang F, & Wang Y. (2021). The N6-methyladenosine RNA-binding protein YTHDF1 modulates the translation of TRAF6 to mediate the intestinal immune response. Nucleic Acids Research, 49(10), 5537-5552.
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