HA-TRB3 and FLAG-β-tubulin constructs have been described elsewhere (5 (link)). Plasmids encoding HA-tagged LRR (leucine-rich-repeat) domains of LRRFIP2 (Addgene plasmid # 21152) and Fli1 (Addgene plasmid # 21151) were a gift from Dr Junying Yuan (Harvard University), and Flag-MyD88 (Addgene plasmid # 13093) was a gift from Dr Ruslan Medzhitov (HHMI, Yale School of Medicine). pcDNA3-HA-Fli1 was generated by PCR-based amplification of full-length, mouse Fli1, using pCMV-Sport6 mFli1 (Horizon Discovery) as template, and cloned into pcDNA3.1 vector using HindIII and Xho1 sites, in frame with a C-terminal HA tag. Myc-APEX2-TRB3 was generated by PCR amplification of APEX2 using pcDNA3-APEX2-NES as template (Addgene plasmid #49386, kind gift of Alice Ting, Stanford University) and subcloned into BamH1 and Xho1 sites of pcDNA3 Myc plasmid, followed by subcloning of TRB3 in frame with APEX2, using EcoRI and XbaI sites. NFκB-luciferase was purchased from Clontech. Nickase-Ninja TRB3 gRNA plasmids were purchased from ATUM Bioscience. Each plasmid expressed CMV promoter driving a Cas9N nickase mutant that causes single-strand breaks and coexpresses two gRNAs using dual U6 promoters. gRNA directed against GFP was included as control (47 (link)). Sequences of dual gRNAs targeting either Exon 2 or Exon 3 of the mouse TRB3 gene are included in Supporting information section.
Pcdna3 apex2 nes
Manufactured by Addgene
Sourced in United States, Germany
The pcDNA3-APEX2-NES is a plasmid vector that expresses the APEX2 enzyme fused with a nuclear export signal (NES). APEX2 is a genetically engineered peroxidase that can be used for proximity-based labeling of proteins in living cells.
Automatically generated - may contain errors
Lab products found in correlation
Nfκb luciferase, by Takara Bio (1 mentions)
Glycine solution, by Merck Group (1 mentions)
Embed 812 resin, by Thermo Fisher Scientific (1 mentions)
3 3 diaminobenzidine (dab), by Merck Group (1 mentions)
Talos l120c, by Thermo Fisher Scientific (1 mentions)
Paav ef1a dio wpre hgh vector, by Addgene (1 mentions)
5 protocols using pcdna3 apex2 nes
1
Generation of TRB3 Gene Knockout Constructs
2
Engineered Flag-APEX2 Fusion Proteins
3
Ultrastructural Imaging of APEX2-TM4SF5 Localization
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Cells were transfected with APEX2‐TM4SF5 plasmid (homemade using pcDNA3 APEX2‐NES [#49386, Addgene, Watertown, MA, USA]) for 48 h, fixed with 2% glutaraldehyde and 2% paraformaldehyde solution at 4°C for 16 h, washed 3 times with 100 mmol/L sodium cacodylate buffer (97068, Sigma‐Aldrich), and treated with 20 mmol/L glycine solution (50046, Sigma‐Aldrich). The cells were then stained with freshly diluted 0.5 mg/mL DAB (D12384, Sigma‐Aldrich) in 10 mmol/L H2O2 solution, washed with sodium cacodylate buffer 3 times, fixed using 2% (W/V) osmium tetroxide (75633, Sigma‐Aldrich) for 40 min on ice, rinsed with ice‐cold distilled water, and dehydrated in a graded ethanol series (50%‐100%) for 15 min at each step. The sample was then mixed 1:1 (v/v) with EMBED‐812 resin (50‐980‐446, Thermo Fisher Scientific) and anhydrous ethanol for 1 h. The mixture was incubated overnight in 2:1 (v/v) resin and then exchanged with 100% resin for 2 h before transfer to fresh resin, followed by polymerization at 60°C for 24 h. Embedded cell pellets were cut with a diamond knife into 50 nm sections and imaged on an FEI‐Tecnai G2 Spirit Bio Twin TEM instrument. Images were taken using a 120 kV transmission electron microscope (Talos L120C, FEI, Hillsboro, OR, USA) at the National Instrumentation Center for Environmental Management (NICEM) in Seoul National University (Seoul, S.
4
Plasmid Toolkit for Cell Signaling Studies
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The following plasmids were used: pCMV6-Myc-DDK-HB-EGF (RC 207688, Origene, Rockville, MD), pCMV6-XL4-AT1R (SC 108918, Origene, Rockville, MD); pEGFR-EGFP (RRID:Addgene_32751, addgene, Watertown, MA; Carter et al. J Biol Chem. 1998 Dec 25;273(52):35000–7), pDsRed2 (#632406, Clontech, Mountain View, CA), pBABEpuro [59 (link)], pcDNA5/FRT/TO-AT1R-APEX (RRID:Addgene_96847, addgene; Paek et al. Cell. 2017 Apr 6;169(2):338–349), pcDNA3 APEX2-NES (RRID:Addgene_49386, addgene).
Unless stated otherwise, all materials were purchased from Sigma, Munich, Germany.
Unless stated otherwise, all materials were purchased from Sigma, Munich, Germany.
5
APEX2-Fused Protein Expression in AAV
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APEX2-NES-P2A-EGFP, LCK-APEX2-P2A-EGFP, and H2B-APEX2-P2A-EGFP were synthesized by Genscript and subcloned into pAAV-EF1a-DIO-WPRE-hGH vector (a gift from Dr. Karl Deisseroth (Addgene plasmid #20297) using restriction enzymes AscI and NheI. APEX2 sequence was cloned based on pcDNA3-APEX2-NES (a gift from Dr. Alice Ting; Addgene plasmid #49386 12 ). APEX AAVs was packaged into adeno-associated virus serotype 1 by the University of North Carolina (UNC) vector core service or Vigene Biosciences (Rockville, MD. USA). AAV8.hSyn.DIO.Cherry was a gift from Dr. Bryan Roth (Cat. No. 50459-AAV8, Addgene).
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