To construct the mKate2-TAPP1-PH, BamHI-mKate2 and TAPP1-PH-Not1 were prepared by PCR with mKate2-P2A-APEX2-TAPP1-PH (Addgene number; #67662) as a template. These PCR products were inserted in pRK5 vector using In-Fusion technique (Takara). pTag-BFP-C-h-Rab5a-c-Myc (#79801), GFP-Rab7a (#61803), LAMP1-mGFP (#34831), and pEGFPN1-human Dynamin K44A (#22197) were obtained from Addgene. FRET-based RhoA biosensor is originally from Dr. Matsuda (Osaka University)39 (link). mCherry-tagged ARAP3 and ARAP3-R982A were generated by fusing red fluorescent protein mCherry into the N-terminal of the ARAP3 templates34 (link). To construct the EGFP-ARAP3-PH(X), each PH domain35 (link) and EGFP sequences were prepared by PCR and inserted in pRK5 vector using In-Fusion technique. VN173-tagged TAPP1 was constructed by inserting PCR fragments of VN173 and TAPP1 PH domain in pRk5 vector. VC155-tagged ARAP3-PH(X) constructs were generated by Xba1/BamH1 replacement of EGFP in EGFP-ARAP3-PH(X) with a VC155 including Xba1/BamH1 site.
Lamp1 mgfp
Manufactured by Addgene
Sourced in United States
LAMP1-mGFP is a plasmid that expresses a fusion protein of the lysosome-associated membrane protein 1 (LAMP1) and monomeric green fluorescent protein (mGFP). It can be used to label lysosomes in live cells.
Automatically generated - may contain errors
Lab products found in correlation
Mapple tomm20 n 10, by Addgene (2 mentions)
Pmxs 3xha egfp omp25, by Addgene (2 mentions)
Pspax2, by Addgene (2 mentions)
Phyper cyto, by Evrogen (2 mentions)
Megfp n1, by Addgene (2 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (2 mentions)
In fusion technique, by Takara Bio (1 mentions)
Gfp rab7a, by Addgene (1 mentions)
Mkate2 p2a apex2 tapp1 ph, by Addgene (1 mentions)
Ptag bfp c h rab5a c myc, by Addgene (1 mentions)
Lenti x concentrator, by Takara Bio (1 mentions)
X tremegene hp, by Roche (1 mentions)
Mrfp lc3, by Addgene (1 mentions)
Egfp lc3, by Genechem (1 mentions)
Plp vsvg, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 3000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Puromycin, by Merck Group (1 mentions)
Nis element ar software, by Nikon (1 mentions)
Lipi red probe ld03 10, by Dojindo Laboratories (1 mentions)
Lysotracker blue dnd 22, by Thermo Fisher Scientific (1 mentions)
11 protocols using lamp1 mgfp
1
Fluorescent Biosensor Cloning Protocol
2
Lentiviral Transduction of Neuronal Constructs
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mApple-TOMM20-N-10 (Addgene; 54955), LAMP1-mGFP (Addgene; 34831), Halo-Parkin WT, Halo-Parkin C431S, and pMXs-3xHA-EGFP-OMP25 (Addgene; 83356) were subcloned into the pER4 lentiviral expression vector using the Nhe I/Not I cut sites. Lentiviral TBC1D15 short hairpin RNA (shRNA; Horizon Discovery; #RHS4533-EG64786) and Scramble shRNA (Horizon Discovery; #RHS6848) were obtained from Horizon Discovery. Lentiviral vectors were transfected into low passage number (< passage 10) human embryonic kidney 293FT cells using X-tremeGENE HP (Roche; 6366546001) together with helper plasmids psPAX2 (Addgene; 12260) and pLP3 (Invitrogen; K497500). Supernatants were collected after 48 hours of expression, and virus was concentrated overnight at 4°C with Lenti-X Concentrator (Clontech; 631232). Quantification of retroviral antigens was determined using a HIV-1 p24 Antigen ELISA Kit (Thermo Fisher Scientific; 22-156-700). Concentrated virus was aliquoted and stored at −80°C for future use. Lentiviral transduction of neurons of all constructs was carried out with an multiplicity of infection (MOI) of 5 for 7 days. Expression of constructs transduced by lentiviral infection was verified by either immunoblot analysis as described above or imaging as described below.
3
Generation of Genetically-Encoded Hyper Probes
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To generate the pHyPer-pexo (referred to as Pexo_Hyper), pHyPer-cyto (FP941; Evrogen) was used as a template. The primer Fw_Hyper_BamHI and Rv_Hyper_PTS1_NotI (Table S1 ) were used to perform PCR. The PCR product was extracted using the clonetech nucleospin gel and PCR cleanup kit (Cat. #740609.5; Takara Clonetech) according to the vendor’s instructions. Both the PCR product and the mEGFP-N1 (Plasmid #54767; Addgene) were digested with BamHI and NotI restriction enzyme and the resulting product was ligated using the DNA Ligation Kit, Mighty Mix (Cat. #6023; Takara Clonetech).
To generate pHyPer-endo (referred to as Endo_Hyper), +HyPer7 (plasmid #136466; Addgene) and the Lamp1-mGFP (plasmid #34831; Addgene) were digested with BamhI and XbaI restriction enzyme. The Hyper7 sequence was then cloned into the Lamp1 backbone by using the DNA Ligation Kit.
To generate pHyPer-endo (referred to as Endo_Hyper), +HyPer7 (plasmid #136466; Addgene) and the Lamp1-mGFP (plasmid #34831; Addgene) were digested with BamhI and XbaI restriction enzyme. The Hyper7 sequence was then cloned into the Lamp1 backbone by using the DNA Ligation Kit.
4
Establishing Stable Cell Lines for mRFP-LC3 and LAMP1-mGFP
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mRFP-LC3 (21075) and LAMP1-mGFP (34831) were obtained from Addgene. EGFP-LC3 and ATG7-siRNA plasmids were constructed by GeneChem Co. LTD (Shanghai, China). Plasmids were transfected into MDA-MB-231 and MCF-7 cells using Lipofectamine 3000 transfection reagent (Invitrogen, L3000). The transfection mixture was removed and replaced with fresh complete medium after 24 h incubation. The sequences of UBC9 shRNA were 5′-TTGGCAGTAAATCGTGTAGGCC-3′ (sh-UBC9–1#) and 5′-ATTTAGAAGTTCCTGTATTCCT-3′ (sh-UBC9–3#).
293FT cells were transfected with pLP1, pLP2, pLP/VSVG (Invitrogen, K4975) and sh-UBC9 or sh-Con plasmid. Supernatants containing the lentivirus were harvested after 48 h incubation and used to infect target cells. MDA-MB-231 and MCF-7 cells were selected with puromycin (4–8 μg/mL) (Sigma, P9620) to establish stable cell lines.
293FT cells were transfected with pLP1, pLP2, pLP/VSVG (Invitrogen, K4975) and sh-UBC9 or sh-Con plasmid. Supernatants containing the lentivirus were harvested after 48 h incubation and used to infect target cells. MDA-MB-231 and MCF-7 cells were selected with puromycin (4–8 μg/mL) (Sigma, P9620) to establish stable cell lines.
5
Visualization of Lipid Droplets and Lysosomes
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To visualize the accumulation of lipid droplets, cells were stained with 1 μM Lipi-Red probe (LD03-10, Dojindo, Kumamoto, Japan) for 30 min. To confirm the colocalization of lipids and lysosomes, the cells were stained with 100 nM Lysotracker Blue DND-22 (L7525, Thermo Fisher Scientific, Waltham, MA, USA) or 100 nM Lysotracker DND-99 (L7528, Thermo Fisher Scientific, Waltham, MA, USA) for 15 min and 1 μM Lipi-Red probe (LD03-10, Dojindo, Kumamoto, Japan) for 30 min. To confirm the location of lysosomes, the hippocampal neurons were transfected with 2 μg of lamp1-mGFP (#34831, Addgene, Watertown, MA, USA) with 2 μL of lipofectamine 2000 reagent (Thermo Fisher Scientific, Waltham, MA, USA). The cells were then stained with anti-GFP antibody (A11122, Invitrogen, Carlsbad, CA, USA). The confocal microscopic images were acquired using an A1 Rsi/Ti-E confocal microscope (Nikon, Tokyo, Japan) with a 60 × oil-immersion lens. Z-stack image sequences were acquired at 0.4 μm intervals and converted with maximal intensity projection using NIS-element AR software (Nikon, Tokyo, Japan).
6
Engineered Organelle Markers for Live-Cell Imaging
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β-actin YFP-Sec61β encodes full-length human ER marker Sec61β, fused to EYFP and was a gift from C. Hoogenraad (Utrecht University, Utrecht, The Netherlands). pB80-KIF1A(1-365)-VVDfast-FLAG-SSPB(micro), encoding opto-kinesin, was derived from pB80-KIF1A(1-365)-VVDfast-GFP-SSPB(micro) (18 (link)) by replacing GFP with a 2xFLAG-tag (DYKDHDGDYKDHD). pB80-LAMP1-mCherry-iLID was cloned in the mammalian expression vector pβactin-16-pl (chicken β-actin promoter, referred to hereafter as pB80) (42 (link)), in which the multiple cloning site was replaced by (Asc I–Xba I–Hind III–Nhe I–Sna BI–Mlu I–Age I). The full-length human lysosome marker LAMP1, without stop codon, was tagged C-terminally with the red fluorescent protein mCherry and the photosensitive heterodimerization module iLID, interspaced by two synthetic 29–amino acid GGGS linkers. The iLID module was derived from pLL7.0-Venus-iLID-Mito (43 (link)), a gift from B. Kuhlman (University of North Carolina at Chapel Hill, NC; Addgene plasmid #60413). Full-length wild-type human LAMP1 was derived from LAMP1-mGFP (44 (link)), a gift from E. Dell’Angelica (University of California, Los Angeles, CA; Addgene plasmid #34831). All constructs were validated by sequencing of the full open reading frame.
7
Plasmid Sourcing and Cloning for Organelle Imaging
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The following plasmids were obtained from Addgene: mApple-TOMM20-N-10 (Addgene; 54955), mCitrine-N1 (Addgene; 54594) (27 (link)), and EBFP2-Lysosomes-20 (Addgene; 55246) were gifts from M. Davidson; LAMP1-mGFP (Addgene; 34831) (28 (link)) was a gift from E. Dell-Angelica; pmTurquoise2-N1 (Addgene; 60561) (29 (link)) was a gift from D. Gadella; Halo-KDEL (Addgene; 124316) (30 (link)) was a gift from J. Wang; pEGFP-parkin WT (Addgene; 45875) (31 (link)) and pEGFP-parkin C431S (Addgene; 45877) (31 (link)) were gifts from E. Fon; EGFP-Rab7A (Addgene; 28047) (32 (link)) was a gift from Q. Zhong; pMXs-3xHA-EGFP-OMP25 (Addgene; 83356) (24 (link)) and pLJC5-Lamp1-RFP-3xHA (Addgene; 102932) (23 (link)) were gifts from D. Sabatini; psPAX2 (Addgene; 12260) was a gift from D. Trono; Lamp1-RFP (Addgene; 1817) (33 (link)) was a gift from W. Mothes; pGEX-4T-3-mR7BD (Addgene; 79149) (34 (link)) was a gift from A. Edinger; and Halo-TOMM20-N-10 (Addgene; 123284) was a gift from K. McGowan. Additional plasmids included the following: EGFP-Rab7 K38R (a gift from P. Song) (9 (link)), pLP3 (Invitrogen; K497500), and pGEX-4T-1 (Millipore Sigma; GE28-9545-49). The following plasmids were generated for this study using standard cloning procedures: LAMP1-Halo, LAMP1-pmTurquoise2, mCitrine-TOMM20, Halo-Parkin WT, Halo-Parkin C431S, pER4-mApple-TOMM20, pER4-LAMP1-mGFP, and pER4-3xHA-EGFP-OMP25.
8
Plasmid Constructs for CHCHD10 and TDP43
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All complementary DNAs (cDNAs) for human CHCHD10WT and variants were synthesized and inserted in the pcDNA3 vector containing a FLAG, HA, or Myc tag by Genescripts. mTagRFP-T-Mito-7 (#58023)64 (link), TDP43tdTOMATO-HA (#28205)65 , EGFP-LC3 (#21073)66 , mCherry-LC3B (#40827)67 (link), pEYFP-C1-DRP1 (#45160)68 (link), MFN2-YFP (#28010)69 (link), pEYFP-N1-PINK1 (#101874)70 , and LAMP1-mGFP (#34831)71 (link) plasmids were obtained from Addgene. FLAG-MFN2WT, FLAG-MFN2S378A, and FLAG-MFN2S378D were described previously51 (link). TARDBPWT, TARDBPG298S, TARDBPA315T, and TARDBPA382T plasmids were gifts from Xinglong Wang. Mito-QC (pBabe.hygro-mcherry-GFP fis 101-152) was provided by Ian Ganley.
9
Generation of Genetically-Encoded Hyper Probes
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To generate the pHyPer-pexo (referred to as Pexo_Hyper), pHyPer-cyto (FP941; Evrogen) was used as a template. The primer Fw_Hyper_BamHI and Rv_Hyper_PTS1_NotI (Table S1 ) were used to perform PCR. The PCR product was extracted using the clonetech nucleospin gel and PCR cleanup kit (Cat. #740609.5; Takara Clonetech) according to the vendor’s instructions. Both the PCR product and the mEGFP-N1 (Plasmid #54767; Addgene) were digested with BamHI and NotI restriction enzyme and the resulting product was ligated using the DNA Ligation Kit, Mighty Mix (Cat. #6023; Takara Clonetech).
To generate pHyPer-endo (referred to as Endo_Hyper), +HyPer7 (plasmid #136466; Addgene) and the Lamp1-mGFP (plasmid #34831; Addgene) were digested with BamhI and XbaI restriction enzyme. The Hyper7 sequence was then cloned into the Lamp1 backbone by using the DNA Ligation Kit.
To generate pHyPer-endo (referred to as Endo_Hyper), +HyPer7 (plasmid #136466; Addgene) and the Lamp1-mGFP (plasmid #34831; Addgene) were digested with BamhI and XbaI restriction enzyme. The Hyper7 sequence was then cloned into the Lamp1 backbone by using the DNA Ligation Kit.
10
Lysosomes Tracking in U2OS Cells
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