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Pfn21a

Manufactured by Promega
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PFN21A is a lab equipment product from Promega. It is a device used for the detection and measurement of protein-protein interactions.

Automatically generated - may contain errors

Lab products found in correlation

Gibson assembly master mix, by New England Biolabs (2 mentions) Ptripz inducible lentiviral vector, by Thermo Fisher Scientific (2 mentions) Gata4 v5, by Promega (2 mentions) Quick ligation kit, by New England Biolabs (2 mentions) In fusion hd cloning kit, by Takara Bio (1 mentions) Q5 site directed mutagenesis kit, by New England Biolabs (1 mentions) Mevalonate, by Merck Group (1 mentions) Bax 2772, by Cell Signaling Technology (1 mentions) Glut1 sc 377228, by Santa Cruz Biotechnology (1 mentions) Protease inhibitor cocktail, by Promega (1 mentions) Magne halotag beads, by Promega (1 mentions) Mammalian lysis buffer, by Promega (1 mentions) Horseradish peroxidase conjugated anti rabbit igg antibody, by GE Healthcare (1 mentions) Fugene hd transfection reagent, by Promega (1 mentions) Pclipf, by New England Biolabs (1 mentions) Psnapf, by New England Biolabs (1 mentions) Pegfp c1, by Takara Bio (1 mentions) Phrl cmv, by Promega (1 mentions) Pfn10a, by Promega (1 mentions) Phrl tk, by Promega (1 mentions)

8 protocols using pfn21a

1

Recombinant ETS1 Constructs Generation

Cited 2 times
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Full-length and truncated ETS1 constructs were amplified from ETS1 cDNA (NM_001143820, Sino Biological, Shanghai, China) and recombined into pNTAP (Agilent Technologies, Santa Clara, CA, USA) and pCMV-3xFlag (Agilent Technologies, Santa Clara, CA, USA) using an In-Fusion HD cloning kit (Clontech, Mountain View, CA, USA). ETS1 T265A/S269A/S273A mutants (ETS1-3A) and ETS1 S272A/S276A mutants (ETS1-2A) were generated by overlapping PCR with the Q5 Site-Directed Mutagenesis Kit (New England Biolabs, Ipswich, MA, USA). Halo-ETS1 and Lenti-ETS1 wild-type and mutant constructs were amplified from pCMV-3xFlag wild-type and mutant ETS1 and recombined into pFN21A (Promega; Halo-ETS1) or pLAS5w.PeGFP-I2-Puro (RNAi core laboratory, Academia Sinica, Taipei, Taiwan; Lenti-ETS1) vectors using the In-Fusion HD cloning kit. GSK3β truncated constructs and MMP9 promoter luciferase constructs have been previously described [31 (link), 46 (link)]. The primers used for DNA constructs are shown in Supplementary Table 1. All constructs were finally confirmed with ABI autosequencer.
Tsai C.L., Jung S.M., Chi L.M., Tsai C.N., Lin C.Y., Chao A, & Lee Y.S. (2021). Glycogen synthase kinase-3 beta (GSK3β)-mediated phosphorylation of ETS1 promotes progression of ovarian carcinoma. Aging (Albany NY), 13(10), 13739-13763.
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2

Generation of Inducible FOXA2, RFP, and FOXA2-CDT1 Clones

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To generate pTRIPZ-FOXA2, RFP, FOXA2-CDT1 clones, pTRIPZ inducible lentiviral vector (Thermo Scientific) and full-length FOXA2 were assembled using Gibson Assembly® Master Mix (NEB). pTRIPZ empty vector was digested with XhoI and MluI to remove shRNAmir regulatory sequences, and digested ends were blunted. The linearized pTRIPZ backbone was digested with BsiWI to generate two fragments, each with one sticky end. The fragments were gel extracted, purified, and ligated using the Quick LigationTM Kit (NEB). Primer sequences are listed in Supplementary Table 1.
To generate HaloTagged-FOXA2 construct, full-length FOXA2 was ligated to pFN21A (Promega)
GATA4-V5 and POU5F1-V5 constructs were obtained from the Broad Institutes Genomics Perturbations platform and are available to purchase through Thermo Fisher.
Donaghey J., Thakurela S., Charlton J., Chen J., Smith Z.D., Gu H., Pop R., Clement K., Stamenova E., Karnik R., Kelley D.R., Gifford C.A., Cacchiarelli D., Rinn J.L., Gnirke A., Ziller M.J, & Meissner A. (2018). Genetic determinants and epigenetic effects of pioneer factor occupancy. Nature genetics, 50(2), 250-258.
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3

Targeting EGFR and Caveolin-1 in Cancer

Cited 1 time
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The target sequences of short-interfering RNAs for EGFR (5' -CAGGAACTGGA TATTCTGAAA-3') 67 (link), Cav1#1 (5'-CACCTTCACTGTGACGAAATA-'3) and Negative (scrambled) Control (5'-AATTCTCCGAACGTGTCACGT-'3) were purchased from Qiagen. Anti-Cav1 (rabbit sc-894, 1:1000 and mouse sc-53564, 1:1000), GLUT1 (sc-377228, 1:1000), GLUT3 (sc-74497, 1:1000), FLOT1 (sc-74566, 1:1000) and β-actin (ACTB, sc-47778, 1:2000) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-total AMPKα (#5831, 1:1000), phospho-AMPKα T172 (#2535, 1:1000) and Bax (#2772, 1:2000) antibodies were purchased from Cell Signaling. Mevalonate (#4667) was purchased from Sigma. HALO-tag expression vectors pFN21A-CAV1 and pFN21A were purchased from Promega. Atorvastatin (ATV; Lipitor) and Simvastatin (Zocor) were purchased from Pfizer and MSD Pharmaceuticals Singapore respectively. Both drugs were dissolved in DMSO for cell culture experiments. For in vivo models, ATV was dissolved in sterile 0.9% NaCl solution.
Ali A., Levantini E., Fhu C.W., Teo J.T., Clohessy J.G., Goggi J.L., Wu C.S., Chen L., Chin T.M, & Tenen D.G. (2019). CAV1 - GLUT3 signaling is important for cellular energy and can be targeted by Atorvastatin in Non-Small Cell Lung Cancer. Theranostics, 9(21), 6157-6174.
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4

Halo- and HA-tagged Protein Purification

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We cloned the Halo- or HA-tagged, full-length WT, R132H or Y208C mutant into the pFN21A (Promega) or pMACS Kk.HA-C vector, respectively. The expression of the Halo- and HA-tagged constructs in the HeLa cells was induced using FuGENE HD Transfection Reagent (Promega), and the transfected cells were grown for 48 h. The cells were then lysed and pulled down using Mammalian Lysis Buffer (Promega) containing Protease Inhibitor Cocktail (Promega) for 15 min, and cellular debris was cleared by centrifugation at 12,000 × g for 10 min. In total, 50 µl of Magne Halo-Tag Beads (Promega) equilibrated with TBS containing 0.05% IGEPAL CA-630 (TBS+) was added to the supernatant. The samples were incubated for 20 min at 22°C with rotation. The supernatant was discarded, and the protein-captured beads were washed thrice with TBS+ and suspended in SDS-PAGE loading buffer. The samples were analyzed by immunoblotting using anti-Halo or anti-HA antibody and horseradish peroxidase-conjugated anti-rabbit IgG antibody (GE Healthcare). The blots were developed using EzWestLumi plus reagents.
Kawakami S., Michishita M., Sakaue M., Morimatsu M., Uemura M., Kashiwagi N., Maeda M., Machida Y., Azakami D., Egusa A.S., Onozawa E., Ishioka K., Watanabe M., Tanaka Y., Omi T, & Ochiai K. (2020). Novel canine isocitrate dehydrogenase 1 mutation Y208C attenuates dimerization ability. Oncology Letters, 20(6), 351.
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5

Plasmid Library for Protein Interactions

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Most plasmids were purchased from Addgene or were shared by independent labs. GFP-tagged proteins candidates were selected based on gene ontology annotation containing terms “identical protein binding” (GO:0042802), “protein homotrimerization” (GO:0070207), “protein trimerization” (GO:0070206), “protein dimerization” (GO:0046983) and “protein tetramerization” (GO:0051262) and independently verified for their self-interacting ability by the tool SLIPPER (http://lidong.ncpsb.org/slipper/index_1.html). The resulting pools of proteins were selected to cover a range of valences. The proteins tested in each class are: monomeric, p53; dimeric, AKT, Rac2; trimeric, HSF1; tetrameric, PKM2; octomeric, PAICS; IDR-containing: FUS, TDP-43. pcDH-Halo-DCP1A, pcDH-SNAP-DCP1A, pcDH-GFP-DCP1A, pcDH-mCherry-DCP1A, and pcDH-CLIP-DCP1A were constructed by first sub-cloning the DCP1A open-reading frame (ORF) from pEGFP-DCP1A into the pcDH backbone to generate pcDH-DCP1A. The ORFs of Halo, SNAP, GFP, mCherry and CLIP were PCR amplified from pFN21A (Promega), pSNAPf (NEB), pEGFP-C1 (Clontech), pEF1a-mCherry (Clontech), and pCLIPf (NEB), respectively. These amplicons were then sub-cloned into the pcDH-DCP1A backbone to generate the appropriate plasmids.
Jalihal A.P., Pitchiaya S., Xiao L., Bawa P., Jiang X., Bedi K., Parolia A., Cieslik M., Ljungman M., Chinnaiyan A.M, & Walter N.G. (2020). Multivalent proteins rapidly and reversibly phase-separate upon osmotic cell volume change. Molecular cell, 79(6), 978-990.e5.
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6

Generation of Inducible FOXA2, RFP, and FOXA2-CDT1 Clones

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To generate pTRIPZ-FOXA2, RFP, FOXA2-CDT1 clones, pTRIPZ inducible lentiviral vector (Thermo Scientific) and full-length FOXA2 were assembled using Gibson Assembly® Master Mix (NEB). pTRIPZ empty vector was digested with XhoI and MluI to remove shRNAmir regulatory sequences, and digested ends were blunted. The linearized pTRIPZ backbone was digested with BsiWI to generate two fragments, each with one sticky end. The fragments were gel extracted, purified, and ligated using the Quick LigationTM Kit (NEB). Primer sequences are listed in Supplementary Table 1.
To generate HaloTagged-FOXA2 construct, full-length FOXA2 was ligated to pFN21A (Promega)
GATA4-V5 and POU5F1-V5 constructs were obtained from the Broad Institutes Genomics Perturbations platform and are available to purchase through Thermo Fisher.
Donaghey J., Thakurela S., Charlton J., Chen J., Smith Z.D., Gu H., Pop R., Clement K., Stamenova E., Karnik R., Kelley D.R., Gifford C.A., Cacchiarelli D., Rinn J.L., Gnirke A., Ziller M.J, & Meissner A. (2018). Genetic determinants and epigenetic effects of pioneer factor occupancy. Nature genetics, 50(2), 250-258.
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7

Plasmid Construction for PXR and PGC1α Studies

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The human PXR (hPXR) pTarget plasmid and p3A4-pGL3 have been reported previously (33 (link)). hPXR-pFN10A was constructed by inserting the amplified hPXR cDNA into pFN10A (Promega) at the SgfI/PmeI sites. The pFN11A-based expression plasmid for the PGC1α-LXXLL motif (EAEEPSLLKKLLLAPANTQ) fused to the GAL4 DBD protein (PGC1α-LXXLL-pFN11A) was constructed previously (51 (link)). phRL-TK, phRL-CMV, and pFN21A were purchased from Promega. All mutations or insertions were generated using PrimeSTAR Max DNA Polymerase (Takara Bio) and confirmed by sequencing.
Shizu R., Nishiguchi H., Tashiro S., Sato T., Sugawara A., Kanno Y., Hosaka T., Sasaki T, & Yoshinari K. (2021). Helix 12 stabilization contributes to basal transcriptional activity of PXR. The Journal of Biological Chemistry, 297(3), 100978.
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8

Generation of Halo-tagged Receptor Constructs

Cited 1 time
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The vector, pFN21A (HaloTag technology, Promega, Madison, WI, USA), encoding the secretory IL-6 signal peptide fused to the N-terminus of Halo-tag was a gift from Dr. Nagase (Kazusa DNA Research Institute). To generate the Halo-PAC1 construct, the hop1 splicing variant of a human PAC1 cDNA was subcloned into the pFN21A vector at SgfI and PmeI restriction sites as described previously (4 (link)). Human 5-HT1A, 5-HT2A, D2 and mGlu2 cDNAs were obtained from the Kazusa Collection of Flexi ORF Clones (Kazusa DNA Research Institute, Chiba, Japan). These clones were also subcloned into the pFN21A vector at SgfI and PmeI restriction sites.
Hayata-Takano A., Shintani Y., Moriguchi K., Encho N., Kitagawa K., Nakazawa T, & Hashimoto H. (2021). PACAP–PAC1 Signaling Regulates Serotonin 2A Receptor Internalization. Frontiers in Endocrinology, 12, 732456.
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