The rat eGFP-KIF1A was a kind gift from Gary Banker (OHSU). The tdTomato-KIF1A plasmid was constructed by restriction site removal of the eGFP from the eGFP-KIF1A plasmid and insertion of tdTomato. The pmTurquoise KIF1A shRNA vector was constructed by restriction site removal of eGFP from the commercially available pSuper vector (Oligoengine) and insertion of the cDNA coding for pmTurquoise (Addgene plasmid #36202). The mCherry-syt-IV and pHluorin-syt-IV were published elsewhere22 (link)30 (link). DsRed2-Mito was purchased from Clontech.
Dsred2 mito
Manufactured by Takara Bio
Sourced in United States
DsRed2-Mito is a fluorescent protein that localizes to the mitochondria of cells. It is derived from the red fluorescent protein DsRed2 and is designed to be targeted to the mitochondria.
Automatically generated - may contain errors
5 protocols using dsred2 mito
1
Molecular Constructs for Kinesin and Synaptic Vesicle Imaging
2
GFP-Sec61-β and DsRed2-Mito Co-Transfection
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Cells under were co‐transfected with GFP‐Sec61‐β (Addgene plasmid #15108) and DsRed2‐Mito (Clontech, #632421) at a 1:1 ratio, using Lipofectamine 2000 (Invitrogen, #11668‐027) in serum‐free DMEM. Twelve hours post‐transfection, cells were analyzed as described (Guardia‐Laguarta et al, 2014 ).
3
Immunolocalization of STING and TBK1 in HeLa Cells
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HeLa cells were seeded on coverslips in 6-well plates the day before transfection and cultured for 24 h. Plasmids of interest were transfected for 36 h before cells were fixed with 4% paraformaldehyde. For immunostaining, cells were first blocked with 5% BSA for 1 h followed by 5-min permeabilization with 0.2% Triton X-100. Cells were then incubated with primary antibody (1:200; mouse anti-HA for STING-β and mouse anti-FLAG for TBK1) overnight at 4°C. After washing with 1× PBS for three times, goat anti-mouse IgG conjugated to TRITC (1:200; Zymed) was added and incubated for 1 h. After washing with 1× PBS for three times, the coverslips were mounted onto the slides for observation using Carl Zeiss LSM 510 META Multiphoton Confocal Microscope. DsRed2-ER and DsRed2-Mito (Clontech) served as ER and mitochondrial markers.
4
Isolating Mitochondria from HeLa DsRed Cells
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We isolated mitochondria from HeLa DsRed (Discosoma sp. red fluorescent protein) 2-mito cells, previously transfected by us with the plasmid DsRed2-Mito (Clontech, Mountain View, CA, USA) [18 (link)]. (Supplementary Materials: Methods ).
5
Stable Mitochondrial Protein Labeling
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The plasmid DsRed2-Mito (Clontech, Mountain View, CA) encodes fusion of Discosoma sp. red fluorescent protein and the mitochondrial targeting sequence (MTS) from subunit VIII of human cytochrome c oxidase, which targets the protein to the mitochondria. HeLa cells were stably transfected with this plasmid according to manufacturer’s manual (HeLa DsRed2-Mito cells, Fig. S1A ), and stability was preserved by selection with G418 (Calbiochem, San Diego, CA, 0.6 mg/ml). The plasmid CMV/myc/mito/GFP (Invitrogen, Cambridge, MA) encodes a fusion of GFP fluorescent protein, and the mitochondrial targeting sequence from subunit VIII of human cytochrome c oxidase. HEK-293 cells were stably transfected with this plasmid according to manufacturer’s manual (HEK-293-GFP-mito cells; see supplemental data Fig. S1B ), and stability was preserved by selection with G418 (1.2 mg/ml).
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