Pmturquoise2 er

Manufactured by Addgene

PmTurquoise2-ER is a plasmid that encodes a fluorescent protein, Turquoise2, targeted to the endoplasmic reticulum (ER) of mammalian cells. Turquoise2 is a variant of the Cyan Fluorescent Protein (CFP) family and exhibits bright fluorescence in the turquoise/blue-green spectrum. The ER-targeting sequence ensures the localization of the fluorescent protein within the ER compartment.

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5 protocols using pmturquoise2 er

Plasmid-Based Viral Gene Expression

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pDZ plasmids contain a bidirectional expression cassette for a given influenza A virus gene segment and have been described previously (45 (link)). pCAGGS-based expression plasmids contain protein-coding sequences under the control of the chicken β-actin promoter (46 (link)). pMD2.G and psPAX2 were a gift from Didier Trono (Addgene; plasmid 12259).
pLVX-IRES-ZsGreen and pLVX-IRES-Puro were purchased from Clontech (respectively, 632187 and 632183). pSpCas9(BB)-2A-GFP (PX458) was a gift from Feng Zhang (Addgene; plasmid 48138) (47 (link)). BiFC expression plasmids for split-YFP were described previously (20 (link)). pmTurquoise2-Mito and pmTurquoise2-ER were a gift from Dorus Gadella (Addgene; plasmids 36208 and 36204, respectively) (48 (link)).

Mazel-Sanchez B., Boal-Carvalho I., Silva F., Dijkman R, & Schmolke M. (2018). H5N1 Influenza A Virus PB1-F2 Relieves HAX-1-Mediated Restriction of Avian Virus Polymerase PA in Human Lung Cells. Journal of Virology, 92(11), e00425-18.

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Plasmid Collection for Organelle Labeling

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These plasmids were a gift from Dorus Gadella: pmTurquoise2-Mito (Addgene plasmid #36208), pmTurquoise2-ER (Addgene plasmid #36204), pmTurquoise2-Peroxi (Addgene plasmid #36203), pmTurquoise2-Golgi (Addgene plasmid #36205) and pmTurquoise2-H2A (Addgene plasmid #36207)^{59 (link)}. pcDNA3.1-MCS-BirA-R118G-HA was a gift from Kyle Roux (Addgene plasmid # 36047)^{55 (link)}. RFP-Dcp1a and RFP-TIA1 were described before^{60 (link)}. MACROD1, MACROD2 and OARD1 (encoding TARG1) were amplified from Hela cDNA using primers with flanking attB sites suitable for the Gateway System (Invitrogen) and inserted into pDONR/zeo with a Gateway BP reaction. pcDNA5/FRT/TO-MACROD1, -MACROD2 or -TARG1 were generated by performing a Gateway LR reaction according to the manufacturer’s instructions. GFP-labelled plasmids were generated by LR reactions into pDEST47 and pDEST53. pcDNA3-mRuby2-MACROD1 and pcDNA3-mRuby2-MACROD1d77 were generated using primers with flanking HindIII and Nhel restriction sites for subsequent insertion into pcDNA3-mRuby2 for which the pcDNA5 constructs were used as PCR template. pcDNA3-BirA-R118G constructs with full length MACROD1, MACROD2 and TARG1 were generated using primers with flanking EcoRI and BamHI restriction sites and the pcDNA5 constructs as template.

Žaja R., Aydin G., Lippok B.E., Feederle R., Lüscher B, & Feijs K.L. (2020). Comparative analysis of MACROD1, MACROD2 and TARG1 expression, localisation and interactome. Scientific Reports, 10, 8286.

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Organelle Visualization and Autophagy Quantification

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Mitochondrial or ER staining was achieved by transient transfection of plasmids pmTurquoise2-Mito (Addgene plasmid #36208) or pmTurquoise2-ER (Addgene plasmid #36204) (gift from Dorus Gadella)^{17 (link)}. These were co-transfected with plasmid mRFP-LC3 (Addgene plasmid #21074; kindly provided by Pr. T. Yoshimori)^{13 (link)}.
TFEB imaging was achieved by transient transfection of plasmids pEGFP-N1-TFEB (gift from Shawn Ferguson) (Addgene plasmid # 38119)^{51 (link)}. Quantitative assessment of the TFEB nuclear localization was achieved by dividing the density of the nuclear signal by the mean density of the fluorescent signal in each cell using the ImageJ software. Fifteen images per condition from five distinct experiments were assessed.
Ad-LC3 imaging was achieved 48 h post infection (MOI1) on INS-1E cells plated on glass cell culture chamber (Ibidi) and photographed on a Leica inverted microscope (×40 oil immersion fluorescence objective). The Ad-LC3 green and red stainings were first converted to a 16 bit format and the signal to noise ratio was determined by applying the Yen threshold method^{52 (link)}. A binary image was then created and the number of particles (LC3 dots) was measured. Data were normalized to the number of cells in each image.

Lambelet M., Terra L.F., Fukaya M., Meyerovich K., Labriola L., Cardozo A.K, & Allagnat F. (2018). Dysfunctional autophagy following exposure to pro-inflammatory cytokines contributes to pancreatic β-cell apoptosis. Cell Death & Disease, 9(2), 96.

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Plasmid Localization and Trafficking

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The expression plasmids pEGFP-hGalectin-3 (GFP-Gal3) and pmRFP-EGFP-Galectin-3 (ptf-Gal3) were purchased from Addgene [deposited by Dr. Tamotsu Yoshimori (Osaka University, Japan)]. The subcellular localization vectors, pmTurquoise2-ER, pmTurquoise2-Golgi, and pmTurquoise2-Peroxi, in which a cyan fluorescence protein (Turquoise) was fused with ER, Golgi, or peroxisome targeting sequences respectively, were obtained from Addgene [deposited by Dr. Dorus Gadella (University of Amsterdam, Netherlands)]. pEGFP-TFEB was obtained from Addgene [deposited by Dr. Shawn Ferguson (Yale Univeristy, USA)]. pLAMP1-GFP and pEGFP-LC3 were kindly provided by Dr. Peter K. Kim (Toronto University, Canada) and Dr. Noboru Mizhushima (Tokyo University, Japan), respectively. pCMV-lyso-pHluorin (Lyso-pHluorin) was purchased from Addgene [deposited by Dr. Christian Rosenmund (Neuroscience Research Center, Germany)]. pEGFP-Ubiquitin (GFP-Ub) and pLAMP1-mCherry were purchased from Addgene [deposited by Dr. Nico Dantuma (Karolinska Institutet, Sweden) and Amy Pamer (University of Colorado, USA)], respectively. Mitochondrial-YFP (pMito-YFP) plasmid was kindly provided by Dr. Gyesoon Yoon (Ajou University, Korea).

Park N.Y., Jo D.S., Kim Y.H., Bae J.E., Kim J.B., Park H.J., Choi J.Y., Lee H.J., Chang J.H., Bunch H., Jeon H.B., Jung Y.K, & Cho D.H. (2021). Triamterene induces autophagic degradation of lysosome by exacerbating lysosomal integrity. Archives of Pharmacal Research, 44(6), 621-631.

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Ubiquitination Compound Library for Drug Screening

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A ubiquitination compound library (L8600) for drug screening was obtained from TargetMol (Boston, MA, USA). VH298 (SML1896) and cycloheximide (01810) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Roxadustat (15294) was obtained from Cayman Chemical Co. (Ann Arbor, MI, USA). VL285 (S0095) was obtained from Selleck Chemicals (Houston, TX, USA). Short interfering RNA (siRNA) targeting NBR1 (5′-GAAGAGGUAUCCAUCAACAUU-3′), p62/SQSTM1 (5′-GCAUUGAAGUUGAUAUCGAUUU-3′), HIF-1α (5’-CAAUCAAGAAGUUGCAUUA-3’), and scrambled siRNA (5′-CCUACGCCACCAAUUUCGU-3′) were synthesized by Genolution (Seoul, Republic of Korea). The subcellular localization vectors, pmTurquoise2-ER, pmTurquoise2-Golgi, pmTurquiose2-Mito, and pmTurquoise2-Peroxi, in which a cyan fluorescence protein (Turquoise) was fused with an ER, Golgi, mitochondria, or peroxisome-targeting sequence, respectively, were obtained from Addgene (36204, 36205, 36208, and 36203; deposited by Dorus Gadella). pcDNA3.1-HA (HA) was obtained from Addgene (128034; deposited by Oskar Laur). HA-tagged ubiquitin WT (HA-UB) was obtained from Addgene (17608; deposited by Ted Dawson).

Kim Y.H., Park N.Y., Jo D.S., Bae J.E., Kim J.B., Park K., Jeong K., Kim P., Yeom E, & Cho D.H. (2024). Inhibition of VHL by VH298 Accelerates Pexophagy by Activation of HIF-1α in HeLa Cells. Molecules, 29(2), 482.

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