MYC-tagged BNIP3L (Myc-Nix), BNIP3L-CYB5 (Nix-CytoB5), BNIP3L-MaoB (Nix-MaoB), BNIP3L-ActA (Nix-ActA), BNIP3L-ΔTM (Nix-delta TM) plasmids (Addgene, 100,795, 100,756, 100,757, 100,758, and 100,755; deposited by Joseph Gordon) were described previously [31 (link)]. The MYC-tagged BNIP3LS212A and BNIP3LS212D were generated using a Q5 site directed mutagenesis kit (New England Biolabs, E0554S). The lentiviral shBNIP3L (Addgene, 100,770; deposited by Joseph Gordon) was generated by ligating oligonucleotides containing the targeting sequence 5ʹ-CAGTTCCTGGGTGGAGCTA-3ʹ into pLKO.1-puro (Addgene, 8453; deposited by Bob Weinberg). The mitochondrial (CMV-mitoCAR-GECO1) and endoplasmic reticulum (CMV-ER-LAR-GECO1) targeted calcium biosensors were gifts from Robert Campbell (Addgene, 46,022 and 61,244; deposited by Robert Campbell) [32 (link),33 (link)]. The shBNIP3L (17,469; deposited by Wafik El-Deiry) [54 (link)], mEmerald-Mito-7 and mCherry-Mito-7 (54,160 and 55,102; deposited by Michael Davidson) [55 ,56 (link)], pPHT-PRKA (60,936; deposited by Anne Marie Quinn) [57 (link)], DAGR (14,865; deposited by Alexandra Newton) [37 (link)], GW1-Mito-pHRed (31,474; deposited by Gary Yellen) [36 (link)] and pcDNA3-FLAG-RHEB (19,996; deposited by Fuyuhiko Tamanoi) [58 (link)] plasmids were purchased from Addgene.
Mcherry mito 7
Manufactured by Addgene
MCherry-Mito-7 is a fluorescent protein construct that localizes to the mitochondria of cells. It is composed of an mCherry fluorescent protein fused to a mitochondrial targeting sequence. This construct can be used to visualize mitochondria in live cells.
Automatically generated - may contain errors
Lab products found in correlation
Killerred dmito, by Evrogen (2 mentions)
Mwasabi mito 7, by Addgene (2 mentions)
Flexiperm midi, by Sarstedt (2 mentions)
Dulbecco s modified eagle s medium, by Merck Group (2 mentions)
Memerald mito 7, by Addgene (1 mentions)
Q5 site directed mutagenesis kit, by New England Biolabs (1 mentions)
Pcdna3 flag rheb, by Addgene (1 mentions)
Shbnip3l, by Addgene (1 mentions)
Gw1 mito phred, by Addgene (1 mentions)
Plko 1 puro, by Addgene (1 mentions)
Pegfp n1, by Takara Bio (1 mentions)
Mcherry2 c1, by Addgene (1 mentions)
One step pcr cloning kit, by Novoprotein (1 mentions)
Lsm 710 laser confocal microscope, by Zeiss (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Fastap phosphatase, by Thermo Fisher Scientific (1 mentions)
Mix2seq, by Eurofins (1 mentions)
Phusion dna polymerase, by Thermo Fisher Scientific (1 mentions)
6 protocols using mcherry mito 7
1
Plasmids for BNIP3L Protein Characterization
2
Overexpression Constructs for Cell Studies
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For overexpression studies 1700011H14Rik/Fame (Dharmacon, MMM1013-202768062), Vangl1-myc (Addgene), Fth1 (Biocat, BC012314-TCM1004-GVO-TRI), pPAmCherry-α-tubulin (Addgene, 31930), mCherry-Mito-7 (Addgene, 55148), were either cloned into pEGFP-N1 (Clontech) or mCherry-expressing vectors (mCherry2-C1, Addgene, 54563). Cloning was done using restriction enzymes. Restriction sites were introduced by PCR primers. For creation of a glycine to alanine (G2A) Fame mutant, respective changes were introduced by primers. Primers were ordered from Sigma Aldrich. Restriction enzymes and ligase were purchased from ThermoFisher. Plasmids were validated by sequencing.
3
Subcellular Localization of Hc-acox-1
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Each of the three variants of Hc-acox-1 and their mutants was cloned into pEGFP-C2 vectors via Hind III and BamH I restriction enzyme sites using a one-step PCR cloning kit (Novoprotein, Shanghai, China) following the supplier’s instructions. HEK293T cells were transiently transfected with the recombinant pEGFP-C2-Hc-acox-1 plasmids with Lipofectamine 2000 (Invitrogen, USA). HEK293T cells were also transfected with mCherry-Mito-7 (Addgene plasmid # 55102) or mCherry-Peroxisomes-2 (Addgene plasmid #54520) plasmids to ascertain localisation of Hc-ACOX-1 to mitochondria or peroxisomes. After 24 hours of incubation, cells were harvested for Western blot analysis and fluorescence observation using a fluorescence microscope (Zeiss, Germany). In brief, cells grown on coverslips were fixed in ice-cold 4% paraformaldehyde in phosphate buffered saline (PBS) (pH 7.4) at 4°C for 15 min and then washed in PBS, followed by permeabilization with 0.1% Triton X-100 in PBS for 5 min. After three washes, cells were incubated with 0.1 μg/mL 4’,6-diamidino-2-phenylindole (DAPI) at 37°C for 15 min and then observed with a Zeiss LSM710 laser confocal microscope (Zeiss, Germany).
4
Plasmid Generation and Validation
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Detailed information regarding the plasmids generated in this study are provided in Supplementary file 5A . Plasmids generated in this study have been produced (i) by ligating annealed primers into BbsI digested plasmids (for cloning of sgRNAs) or (ii) by ligating digested PCR-amplified cDNAs into dephosphorylated plasmids. Phusion DNA polymerase, FastDigest restrictions enzymes and FastAP phosphatase were used (Thermo Fisher Scientific). For all plasmids, DNA sequencing (Mix2Seq, Eurofins Genomics) was used with the specified primers, to confirm that the desired sequence was inserted. All new plasmids have been deposited on Addgene. The plasmids ERmox-GFP (Addgene #68072, Costantini et al., 2015 (link)), Ub-G76V-YFP (Addgene #11949, Menéndez-Benito et al., 2005 (link)), mCherry-Mito-7 (Addgene #55102, Olenych et al., 2007 (link)), pEGFP-N1-ATG-FLAGC (Addgene #60360, Britton et al., 2014 (link)), pEGFP-C1-FLAGN (Addgene #46956, Britton et al., 2013 (link)), pICE-EGFP-FLAG-Ku70siR-WT (Addgene #46961, Britton et al., 2013 (link)), and pCAG-eSpCas9-2A-GFP (Addgene #79145) were provided by Addgene thanks to Addgene contributors.
5
Plasmid Delivery into Cultured CGNs
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To introduce plasmids into cultured CGNs, either electroporation or the calcium phosphate method was used. Electroporation was performed as previously described (33) . CGNs (3 × 10 6 cells) and plasmids were suspended in 70 µl of Dulbecco's modified Eagle's medium (Sigma-Aldrich) and exposed to decay pulses using a CUY21-edit II pulse generator (BEX, Tokyo, Japan) as follows: poring pulse, 275 V for 1 ms; driving pulse,+ 20 V for 50 ms × 5 times at 50 ms intervals with reversal of polarity. In the experiment shown in Fig. 5, 1.2 µg of KillerRed-dMito (Evrogen, Moscow, Russia) or mCherry-Mito-7 (Addgene #55102) (34) together with 2.8 µg of mWasabi-Mito-7 (Addgene #56508) (35) and 0.5 µg of bcl-xl/pcDNA3 (20) was introduced. Immediately after electroporation, prewarmed media was added to the cell, and 5 × 10 5 cells were spread in the grass bottom plate attached with flexiPERM midi (Sarstedt). The calcium phosphate method was performed as previously described (32) .
6
Plasmid Delivery into Cultured CGNs
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To introduce plasmids into cultured CGNs, either electroporation or the calcium phosphate method was used. Electroporation was performed as previously described (33) . CGNs (3 × 10 6 cells) and plasmids were suspended in 70 µl of Dulbecco's modified Eagle's medium (Sigma-Aldrich) and exposed to decay pulses using a CUY21-edit II pulse generator (BEX, Tokyo, Japan) as follows: poring pulse, 275 V for 1 ms; driving pulse,+ 20 V for 50 ms × 5 times at 50 ms intervals with reversal of polarity. In the experiment shown in Fig. 5, 1.2 µg of KillerRed-dMito (Evrogen, Moscow, Russia) or mCherry-Mito-7 (Addgene #55102) (34) together with 2.8 µg of mWasabi-Mito-7 (Addgene #56508) (35) and 0.5 µg of bcl-xl/pcDNA3 (20) was introduced. Immediately after electroporation, prewarmed media was added to the cell, and 5 × 10 5 cells were spread in the grass bottom plate attached with flexiPERM midi (Sarstedt). The calcium phosphate method was performed as previously described (32) .
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