AML12 cells were transfected with pEGFP-N1-TFEB (38119; Addgene) using lipofectamine 2000 (11668019; Invitrogen) according to the manufacturer’s instructions. After transfection, cells were incubated in medium containing 1000 ug/mL G418 (10131; Gibco) for clonal selection. GFP-TFEB positive clone cells were expanded for cellular GFP-TFEB location studies. Fluorescence images were acquired under a fluorescence microscope (Nikon Eclipse 200) with MetaMorph software.
Pegfp n1 tfeb
Manufactured by Addgene
Sourced in Japan, United States
PEGFP-N1-TFEB is a plasmid vector that contains the coding sequence for the transcription factor EB (TFEB) fused to the green fluorescent protein (GFP). This plasmid can be used for the expression and visualization of the TFEB protein in mammalian cell lines.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 3000, by Thermo Fisher Scientific (4 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Prk5 ha gst ragc 75l, by Addgene (2 mentions)
Fugene 6 reagent, by Promega (2 mentions)
Eclipse 200, by Nikon (1 mentions)
Lysotracker green dnd 26, by Thermo Fisher Scientific (1 mentions)
Doxorubicin, by Selleck Chemicals (1 mentions)
Pmxs puro gfp p62, by Addgene (1 mentions)
Hoechst dye, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 488 labeled anti mouse, by Thermo Fisher Scientific (1 mentions)
Inverted microscope, by Leica camera (1 mentions)
Pmturquoise2 mito, by Addgene (1 mentions)
Pmturquoise2 er, by Addgene (1 mentions)
Mrfp lc3, by Addgene (1 mentions)
Pegfp c1 plasmid vector, by Takara Bio (1 mentions)
Hoechst 33342, by Bio-Rad (1 mentions)
Purelinktm hipure plasmid midiprep kit, by Thermo Fisher Scientific (1 mentions)
Competent high dh5α, by Toyobo (1 mentions)
Lipofectaminetm 3000 reagent, by Thermo Fisher Scientific (1 mentions)
Pbabe puro mcherry egfp lc3b, by Addgene (1 mentions)
10 protocols using pegfp n1 tfeb
1
Overexpression of GFP-TFEB in AML12 Cells
2
Doxorubicin and Autophagy Modulation
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Doxorubicin was from Selleckchem (Houston, TX). HPβCD was from Cayman Chemical Company (Ann Arbor, MI). 10-(4′-(N-diethylamino)butyl)-2-chloro-phenoxazine hydrochloride (10-NCP) was from Calbiochem (San Diego, CA). NH4Cl was from Biomatik (Wilmington, DE). Lysotracker Green DND-26 was from Life Technologies (Carlsbad, CA). Antibodies against actin (#3700, 1:1.000) and p62 (#5114, 1:1.000) were from Cell Signaling (Danvers, MA). Antibody against tubulin (#ab7751, 1:1.000) was from Abcam (Cambridge, MA). Antibody against MAP2c (#sc-74421, 1:100) was from Santa Cruz Biotechnologies (Dallas, TX), and antibody against LC3 (#PD014, 1:1.000) was from MBL (Woburn, MA). Anti-mouse Alexa Fluor 488-labeled (#A11001, 1:200) was from Life Technologies. Hoechst dye was from Santa Cruz Biotechnology. pEGFP-N1-TFEB was from Addgene (#38119, deposited by Dr. Shawn Ferguson, Yale School of Medicine). pGW1-beclin1-GFP was described [37 (link)]. pGW1-GFP-p62 was cloned from pMXs-puro GFP-p62, which was from Addgene (#38277, deposited by Dr. Noboru Mizushima, the University of Tokyo).
3
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 method52 (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.
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 method52 (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.
4
Generating tfLC3 Vector for Protein Study
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To generate tfLC3 vector, sequences encoding mCherry and human LC3B were inserted into the pEGFP-C1 plasmid vector (Clontech, 6084–1). pEGFP-N1-TFEB (38119, Dr. Shawn Ferguson’s lab) was purchased from Addgene. EGFP-tagged HTT exon1 (pEGFP-Q23 or pEGFP-Q74) has been described previously [10 (link)]. Transfections were performed with Lipofectamine 3000 (Invitrogen, L3000008) according to the manufacturer’s protocol unless otherwise stated.
5
TFEB Nuclear Translocation Assay with AR-A014418
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pEGFP-N1-TFEB (plasmid#38119) was purchased from Addgene, Inc. The plasmid was transformed into competent high DH5α (TOYOBO Co., Ltd., Osaka, Japan, DNA-903), and plasmid DNA was extracted and purified using PureLinkTM HiPure Plasmid Midiprep Kit (Invitrogen; Thermo Fisher Scientific, Inc.).
The cells (70–90% confluent) were grown in 24-well plates and transfected with the plasmid according to the LipofectamineTM 3000 Reagent protocols. After 24 h of incubation, the cells were treated with 10 µM AR-A014418 for 24 h, and nuclear translocation of TFEB-EGFP was examined and analyzed via fluorescent microscopy (Olympus, IX71) at 40× magnification. The absorption filters used were BA510IF for green fluorescence. The nuclei were stained with Hoechst 33342 (Bio-Rad Laboratories, Inc., 1351304) 5 µg/mL for 10 min at room temperature.
The cells (70–90% confluent) were grown in 24-well plates and transfected with the plasmid according to the LipofectamineTM 3000 Reagent protocols. After 24 h of incubation, the cells were treated with 10 µM AR-A014418 for 24 h, and nuclear translocation of TFEB-EGFP was examined and analyzed via fluorescent microscopy (Olympus, IX71) at 40× magnification. The absorption filters used were BA510IF for green fluorescence. The nuclei were stained with Hoechst 33342 (Bio-Rad Laboratories, Inc., 1351304) 5 µg/mL for 10 min at room temperature.
6
Transfection and Gene Knockdown Assays
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pEGFP-N1-TFEB, pBABE-puro mCherry-EGFP-LC3B, α-Syn-A53T, and pCAX-APP-Swe/Ind were purchased from Addgene (Watertown, MA, USA) and were transfected into cells via Lipofectamine™ 2000 (Invitrogen) in accordance with the manufacturer's protocol. To knockdown the genes of p38, Perk and Tfeb, SH-SY5Y cells were transfected with scramble siRNA (scRNA) (Ambion, Austin, TX, USA), as the control siRNA, p38 (Cell Signaling), Perk and Tfeb siRNA (Santa Cruz Biotechnology, CA, USA) using Lipofectamine™ 2000 (Invitrogen) method according to the manufacturer’s protocol. After 36 h, cells were treated with indicated drugs.
7
Transfection and Silencing of PEX5 and TFEB
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Cells were cultured in 60 mm culture plates until they reached ~50–60% confluence. They were then transfected with 100 nM control siRNA, PEX5 siRNA (#1 sense-CGUAUCCUGGGAUCUCUCU, antisense-AGAGAGAUCCCAGGAUACG, #2 sense-GAAUUCAUCUCUGAAGUUA, antisense-UAACUUCAGAGAGAUGAAUUC-3, Bioneer, Daejeon, Korea), or TFEB siRNA (sc38509, Santa Cruz Biotechnology) for 24 h in serum-free medium, using Lipofectamine RNAiMax transfection reagent (Invitrogen/Life Technologies), and then exposed to serum starvation for 24 h. The effect of gene silencing was determined by Q-PCR and western blot analysis. For plasmid transfection, cells were transiently transfected with GFP-LC323 (link), pEGFP-N1, pEGFP-N1-TFEB (Addgene, # 38119), or TFEB-S142A plasmids by using Lipofectamine 3000 (Invitrogen/Life Technologies), according to the manufacturer’s instructions.
8
Cloning and expression of human FNIP1
9
Plasmid Transfection for TFEB Regulation
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The following plasmids from Addgene were used: CI RagA (pRK5 HA-RagA T21N) #99711, CI RagC (pRK5 HA-RagC Q120L) #99720 and pEGFP-N1-TFEB (Addgene #38119). TFEB-GFP wild-type and delta30 mutants were developed by Dr. Shawn Ferguson23 (link). The GPNMB reporter activity plasmid was previously described25 (link). Plasmids were expressed using Fugene6 reagent (Promega) for immunofluorescence or Lipofectamine 3000 (Invitrogen) for immunoblotting.
10
Plasmid Transfection Optimization for HeLa, HEK293T Cells
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The following plasmids were used in HeLa-TFEB-GFP cells: pRK5 HA- RAGA (#99710), pRK5-HA GST RAGA 66L (#19300), pRK5 HA- RAGC (#99718), and pRK5-HA GST RAGC 75L (#19305), were purchased from Addgene. The following plasmids were used in HeLa cells: TFEB-GFP wild type, TFEB-GFP S142A, TFEB-GFP S211A, TFEB-GFP S142A/S211A developed by Dr. Shawn Ferguson. The following plasmids were used in HEK293T cells: pEGFP-N1-TFEB (Addgene #38119), pCMV-Tag3B vector plasmid (Agilent) and pCMV-Tag3B expressing myc-FLCN plasmid57 (link). All plasmids were expressed using Fugene6 reagent (Promega) for 48 h in immunofluorescence experiments and using Lipofectamine 3000 (Invitrogen) for 6 h in immunoblotting experiments.
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