For mammalian expression, cDNAs encoding ATG5, LAMP1 (human), LC3B (human), MOAP1 (human full-length or the indicated mutants), MTCH2 (human) and STX17 (human) were cloned into the pXJ-40 expression vector (Life Science Market, PVT20255). pBMN HA-GBRP, pMX-IY HA-GBRPL1 and pMX-IY HA-GBRPL2 was a gift from Michael Lazarou (Addgene, 89296, 89297 and 89298) [15 (link)]. pEGFP-2xFYVE was a gift from Harald Stenmark (Addgene, 140047) [48 (link)]. pX330-U6-Chimeric_BB-CBh-hSpCas9 was a gift from Dr Feng Zhang (Addgene, 42230) [49 (link)]. pMRX-IP-GFP-LC3-RFP-LC3ΔG was a gift from Dr. Noboru Mizushima (Addgene, 84572) [32 (link)].
Pmrx ip gfp lc3 rfp lc3δg
Manufactured by Addgene
Sourced in United States
PMRX-IP-GFP-LC3-RFP-LC3ΔG is a plasmid that expresses a fusion protein consisting of GFP-LC3 and RFP-LC3ΔG. It is designed for the study of autophagy.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Px330 u6 chimeric bb cbh hspcas9, by Addgene (1 mentions)
Neb stable competent e coli, by New England Biolabs (1 mentions)
Purelinktm hipure plasmid midiprep kit, by Thermo Fisher Scientific (1 mentions)
Anti gfp sc 9996, by Santa Cruz Biotechnology (1 mentions)
Lsm800 confocal microscope, by Zeiss (1 mentions)
Anti rfp 600 401 379, by Rockland Immunochemicals (1 mentions)
Prolong gold antifade mounting media, by Thermo Fisher Scientific (1 mentions)
Pegfp n1, by Takara Bio (1 mentions)
Pegfp c1, by Takara Bio (1 mentions)
Pcdna3.1 ha, by Thermo Fisher Scientific (1 mentions)
Pmcherry n1, by Takara Bio (1 mentions)
Pbabepuro gfp lc3, by Addgene (1 mentions)
P3xflag myc cmv 24, by Merck Group (1 mentions)
Human fetal brain cdna library, by Takara Bio (1 mentions)
8 protocols using pmrx ip gfp lc3 rfp lc3δg
1
Mammalian Expression Vector Construction
2
Autophagic Flux Measurement in Cell Lines
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PC3 and DU145 cells were stably transfected with pMRX-IP-GFP-LC3-RFP-LC3ΔG (Addgene #84572), and single-cell clones were validated to avoid homologous recombination between the two LC3 fragments during retrovirus infection. For autophagic flux detection, 10,000 cells were plated in 96-well plates and incubated with various compounds in complete medium for 24 hours. GFP and RFP intensities were measured on a TECAN M1000 plate reader.
3
Monitoring Autophagy Using pMRX-IP-GFP-LC3-RFP-LC3ΔG Plasmid
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pMRX-IP-GFP-LC3-RFP-LC3ΔG (plasmid # 84572) was purchased from Addgene, Inc. (Watertown, MA, USA). The plasmid was transformed into NEB® stable Competent E. coli (New England Biolabs, Inc., Ipswich, MA, USA, C3040H), and plasmid DNA was extracted and purified using PureLinkTM HiPure Plasmid Midiprep Kit (Invitrogen; Thermo Fisher Scientific, Inc., Carlsbad, CA, USA).
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 12, 24, and 48 h, and images were captured with fluorescence microscope (Olympus, Tokyo, Japan, IX71) at 40× magnification. The absorption filters used were BA510IF for green fluorescence and BA575IF for red fluorescence. The fluorescence intensity was quantified using the ImageJ imaging software program (version 1.53t).
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 12, 24, and 48 h, and images were captured with fluorescence microscope (Olympus, Tokyo, Japan, IX71) at 40× magnification. The absorption filters used were BA510IF for green fluorescence and BA575IF for red fluorescence. The fluorescence intensity was quantified using the ImageJ imaging software program (version 1.53t).
4
Visualizing Autophagy in Mouse Corneal Cells
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Mouse corneal endothelial cell (MCEC) WT and KO cells were grown on glass coverslips. Using Lipofectamine 3000 (L3000001; Thermo Fisher Scientific), following manufacturer's instructions, cells were transfected with pMRX-IP-GFP-LC3-RFP-LC3ΔG (#84572; Addgene). Then, 72 hours post transfection, the cells were subject to treatment in assay media for 16 hours. Following this, the cells were fixed with 4% paraformaldehyde for 5 minutes, permeabilized using 0.1% Triton X-100 for 5 minutes, and blocked using 3% BSA for 1 hour. Primary antibody incubation using 1:100 anti-GFP (#sc-9996; Santa Cruz Biotechnology, Dallas, TX, USA) and 1:100 anti-RFP (#600-401-379; Rockland Immunochemicals, Gilbertsville, PA, USA) was conducted over night at 4°C. Following secondary antibody incubation, and washes, the cells were mounted using prolong gold antifade mounting media (P36930; Thermo Fisher Scientific) and examined by Zeiss LSM 800 confocal microscope (Zeiss, Oberkochen, Germany) using 63X oil objective.
5
Autophagic Flux Measurement in Cell Lines
6
Plasmid Constructs for Cell Biology
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pEGFP-C1 (Clonetech, PT32595), pEGFP-N1 (Clonetech, PT3027-5), pmCherry-N1 (Clonetech, PT3974-5), pCDNA5-FRT-TO-3 × FLAG (Invitrogen, V6010-20), pcDNA3.1-HA (Invitrogen, V709-20), pmCherry-EGFP (Addgene, 86639; Ivan Yudushkin lab), pOG44 (Stratagene, 1141), pMRX-IP-GFP-LC3-RFP-LC3ΔG (Addgene, 84572; Noboru Mizushima lab).
7
Retroviral Expression of GFP-LC3 and RFP-LC3ΔG
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Recombinant plasmid for retroviral expression of GFP‐tagged LC3 (pBABEpuro GFP‐LC3; Addgene plasmid number 22405) was created by Dr Jayanta Debnath.20 Another retroviral vector expressing GFP‐LC3‐RFP‐LC3ΔG fluorescence probe (pMRX‐IP‐GFP‐LC3‐RFP‐LC3ΔG; Addgene plasmid number 84572) was constructed by Dr Noboru Mizushima.21 Both plasmids were obtained from Addgene. Generation of retrovirus and transduction of experimental cell lines were performed following standard protocol.
Chemically synthesized shRNA sequences targeting Atg7,20 , 22 Bak23 and scrambled control (http://www.hollingscancercenter.org/research/shared-resources/shRNA/Vectors.pdf ) were procured from Integrated DNA Technologies, Inc. The sequences were annealed and sub‐cloned into the AgeI and EcoRI sites of the pLKO.1 puro vector. Standard protocol was followed for generation of lentivirus and transduction of cells.
Cells, transduced with retro/lentiviruses encoding desired genes/shRNAs, were cultured in growth media as mentioned above. Puromycin was added into the media after 48 hours of transduction. After maintaining the cells for ~10 days in puromycin‐containing media, viable stable clones were propagated and used for experiments.
Chemically synthesized shRNA sequences targeting Atg7,
Cells, transduced with retro/lentiviruses encoding desired genes/shRNAs, were cultured in growth media as mentioned above. Puromycin was added into the media after 48 hours of transduction. After maintaining the cells for ~10 days in puromycin‐containing media, viable stable clones were propagated and used for experiments.
8
Plasmid Construction and Characterization
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GFP, GFP-TFEB, GFP-BCL2, GFP-LC3, GFP-Htt-60Q, GFP-Htt-150Q, GFP-BCL2, FLAG, FLAG-BECN1, mCherry, mCherry-GFP-LC3B, GFP-GAggagca (GA∗30), GFP-GRggaaga (GR∗30), GFP-PRccaaga (PR∗30), Flag-GAggagca (GA∗30), Flag-GRggaaga (GR∗30) and mCherry-PRccccgg (PR∗46) plasmids were described previously11 (link),17 (link),20 , 21 (link), 22 (link), 23 (link), 24 (link). mCherry-LC3B was a gift from Dr. David Rubinsztein (Addgene plasmid #40827). pMRX-IP-GFP-LC3-RFP-LC3ΔG was a gift from Dr. Mizushima (Addgene plasmid #84572). ATG5-GFP, ATG9-GFP, DFCP1-GFP and ATG16-mCherry were kindly provided by Dr. Quanhong Ma (Soochow University, China). GABARAPL1 cDNA was first amplified using PCR from a human fetal brain cDNA library (Clontech) using the primers 5′-GAAGATCTACCATGAAGTTCGTGTAC-3′ and 5′-GCGTCGACTCACAGACCGTAGAC-3′. The PCR product was subsequently inserted into the p3xFLAG-Myc-CMV-24 (Sigma) vector at the Bgl II/Sal I sites.
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