other:Article Title: Neuronal ER–plasma membrane junctions organized by Kv2–VAP pairing recruit Nir proteins and affect phosphoinositide homeostasis
Article Snippet: RRID:AB_2722711 Tissue cultured supernatant (1:5) 6 Nir2 Recombinant fragment of human Nir2 aa 589–651 Rabbit pAb, Abcam catalog no. ab221381, RRID:AB_2801563 10 μg/ml ( A ), 1 μg/ml ( E ) S1 Nir2 Recombinant peptide of human Nir2 aa 365–444 Rabbit pAb, Novus Biologicals catalog no. NBP2–34132, RRID: AB_2801564 10 μg/ml ( A ) 6, S1 RyRs (34C) Partially purified chicken pectoral muscle ryanodine receptor Mouse IgG1 mAb, Developmental Studies Hybridoma Bank, RRID: AB_528457 1 μg/ml 6 Open in a separate window Antibody information.
Generated:Article Title: Differential expression levels of Sox9 in early neocortical radial glial cells regulate the decision between stem cell maintenance and differentiation
Article Snippet: Hes5p-dsRed (Addgene plasmid #26868) was generated by Nicholas Gaiano ( ). .. Hes5p-GFP was generated by replacing dsRed2 with eGFP in Hes5p-DsRed ( ). pCAG-mRFP1 (Addgene plasmid #32600) was deposited by Anna-Katerina Hadjantonakis ( ). pCAG-mRFP;Hes5p-GFP was generated by subcloning pCAG-mRFP and a poly-adenylation signal into Hes5p-GFP. pCAG-CreERT2 (Addgene plasmid #14797) was deposited by Connie Cepko ( ). .. Hes5p-CreERT2 was generated by replacing pCAG with Hes5p. pCIG (Addgene plasmid #11159) was deposited by Connie Cepko ( ).
Plasmid Preparation:Article Title: Differential expression levels of Sox9 in early neocortical radial glial cells regulate the decision between stem cell maintenance and differentiation
Article Snippet: Hes5p-dsRed (Addgene plasmid #26868) was generated by Nicholas Gaiano ( ). .. Hes5p-GFP was generated by replacing dsRed2 with eGFP in Hes5p-DsRed ( ). pCAG-mRFP1 (Addgene plasmid #32600) was deposited by Anna-Katerina Hadjantonakis ( ). pCAG-mRFP;Hes5p-GFP was generated by subcloning pCAG-mRFP and a poly-adenylation signal into Hes5p-GFP. pCAG-CreERT2 (Addgene plasmid #14797) was deposited by Connie Cepko ( ). .. Hes5p-CreERT2 was generated by replacing pCAG with Hes5p. pCIG (Addgene plasmid #11159) was deposited by Connie Cepko ( ).
Article Title: Local Electric Field Controls Fluorescence Quantum Yield of Red and Far-Red Fluorescent Proteins
Article Snippet: .. DsRed2 and mPlum were encoded on pBAD; DsRed2-pBAD was a gift from Michael Davidson (Addgene plasmid # 54608; http://n2t.net/addgene:54608 ; RRID:Addgene_54608), and mPlum-pBAD was a gift from Michael Davidson and Roger Tsien (Addgene plasmid # 54564; http://n2t.net/addgene:54564 ; RRID:Addgene_54564). .. DsRed2 and mPlum were encoded on pBAD; DsRed2-pBAD was a gift from Michael Davidson (Addgene plasmid # 54608; http://n2t.net/addgene:54608 ; RRID:Addgene_54608), and mPlum-pBAD was a gift from Michael Davidson and Roger Tsien (Addgene plasmid # 54564; http://n2t.net/addgene:54564 ; RRID:Addgene_54564).
Article Title: Mitochondrial Calcium Waves by Electrical Stimulation in Cultured Hippocampal Neurons.
Article Snippet: Mitochondria are critical to cellular Ca2+ homeostasis via the sequestering of cytosolic Ca2+ in the mitochondrial matrix.. Mitochondrial Ca2+ buffering regulates neuronal activity and neuronal death by shaping cytosolic and presynaptic Ca2+ or controlling energy metabolism.. Dysfunction in mitochondrial Ca2+ buffering has been implicated in psychological and neurological disorders.
Article Title: The DCC receptor regulates astroglial development essential for telencephalic morphogenesis and corpus callosum formation
Article Snippet: .. To generate the pCAG-DCC:tdTomato plasmid, DCC:TDTOMATO (pmDCC:TDTOMATO; provided by Erik Dent, University of Wisconsin-Madison) was subcloned into the pCag-DsRed2 plasmid (Addgene, 15777, Cambridge, MA), by excising DsRed2. .. For site-directed mutagenesis, the QuickChange II Site-Directed Mutagenesis Kit (Stratagene, Catalogue #200524) was used in accordance with the manufacturer’s instructions.
Article Title: Impaired AMPARs Translocation into Dendritic Spines with Motor Skill Learning in the Fragile X Mouse Model
Article Snippet: .. For the morphologic marker, we excised DsRed2 from addgene Plasmid #11151 using the KpnI and NotI restriction sites and cloned tdTomato into the vector under the CAG promoter using In-Fusion HD Cloning kits (Clonetech Takara Bio, catalog #639649). ..
Article Title: NIX initiates mitochondrial fragmentation via DRP1 to drive epidermal differentiation
Article Snippet: The retroviral plasmid pCLBW-Cox8-GFP-mCh (Addgene plasmid #78520) ( ) was used to express the tandem fluorophores EGFP-mCherry linked to the C terminus of the mitochondrial targeting sequence of Cox8. .. All of the remaining retroviral plasmids were constructed by inserting the following tagged protein sequences into the multi-cloning site of the pLZRS retroviral vector (a gift from Dr. Kathleen Green Lab, Northwestern Univ., Chicago, IL): ( 1 ) Mito-dsRed (a gift from Dr. Thomas Schwarz Lab, Harvard Univ., Boston, MA) links the dsRed2 fluorophore to the C terminus of the Cox8 mitochondrial targeting sequence; ( 2 ) TOM20-mCh (from Addgene plasmid #55146 from Dr. Michael Davidson Lab, Florida State Univ., Tallahassee, FL) links mCherry to the C terminus of full-length human TOM20; ( 3 ) GFP-NIX was made by linking EGFP to the N terminus of the full-length human BNIP3L (NIX) AA 1–219 (from Addgene plasmid #17467) ( ) with an intervening linker sequence of 5′-SDLELKL-3′; ( 4 ) GFP-NIX-WA was made by PCR-based site-directed mutagenesis of GFP-NIX to change tryptophan 36 to an alanine residue (W36A); ( 5 ) GFP-NIX-2GA was made by PCR-based site-directed mutagenesis of GFP-NIX to change glycines 204 and 208 to alanine residues (G204A/G208A); ( 6 ) GFP-Mito was made by linking EGFP to the N terminus of AA 186–219 from full-length human BNIP3L (NIX) with an intervening linker sequence of 5′-SDLELKL-3′; ( 7 ) Tubulin-mCh (a gift from Dr. Kathleen Green Lab, Northwestern Univ., Chicago, IL) links the mCherry fluorophore to the C terminus of mouse beta-tubulin; ( 8 ) SNAP-DRP1-K38A (from Addgene plasmid #45161) ( ) links the SNAPf tag to the N terminus of human DRP1 containing a point mutation of lysine 38 to alanine (K38A); ( 9 ) Mito-SNAP was made from Mito-dsRed by replacing dsRed2 with the SNAPf tag at the C terminus of the Cox8 mitochondrial targeting sequence; and ( 10 ) Halo-FIS1 (from Addgene plasmid #111136) ( ) links the Halo7 tag to the N terminus of human FIS1. .. Bafilomycin A1 (BafA1), 100 μM (Sigma #B1793); carbonyl cyanide 3-chlorophenylhydrazone (CCCP), 10 μM (Sigma #C2759); cobalt chloride (CoCl 2 ), 100 μM (Sigma #15862); deferiprone (DFP), 1 mM (Sigma #Y0001976); dimethyloxalylglycine (DMOG), 1 μM (Sigma #D3695); ethyl(2-(5-nitrothiophene-2-carboxamido)thiophene-3-carbonyl)carbamate (EACC), 10 μM (AOBIOUS #AOB13386); EUK134, 50 μM (Sigma #SML0743); Mdivi-1, 25 μM (Cell Signaling #CAS-338967–87-6); MG132, 10 μM (Sigma #M8699).
Subcloning:Article Title: Differential expression levels of Sox9 in early neocortical radial glial cells regulate the decision between stem cell maintenance and differentiation
Article Snippet: Hes5p-dsRed (Addgene plasmid #26868) was generated by Nicholas Gaiano ( ). .. Hes5p-GFP was generated by replacing dsRed2 with eGFP in Hes5p-DsRed ( ). pCAG-mRFP1 (Addgene plasmid #32600) was deposited by Anna-Katerina Hadjantonakis ( ). pCAG-mRFP;Hes5p-GFP was generated by subcloning pCAG-mRFP and a poly-adenylation signal into Hes5p-GFP. pCAG-CreERT2 (Addgene plasmid #14797) was deposited by Connie Cepko ( ). .. Hes5p-CreERT2 was generated by replacing pCAG with Hes5p. pCIG (Addgene plasmid #11159) was deposited by Connie Cepko ( ).
Droplet Countercurrent Chromatography:Article Title: The DCC receptor regulates astroglial development essential for telencephalic morphogenesis and corpus callosum formation
Article Snippet: .. To generate the pCAG-DCC:tdTomato plasmid, DCC:TDTOMATO (pmDCC:TDTOMATO; provided by Erik Dent, University of Wisconsin-Madison) was subcloned into the pCag-DsRed2 plasmid (Addgene, 15777, Cambridge, MA), by excising DsRed2. .. For site-directed mutagenesis, the QuickChange II Site-Directed Mutagenesis Kit (Stratagene, Catalogue #200524) was used in accordance with the manufacturer’s instructions.
Marker:Article Title: Impaired AMPARs Translocation into Dendritic Spines with Motor Skill Learning in the Fragile X Mouse Model
Article Snippet: .. For the morphologic marker, we excised DsRed2 from addgene Plasmid #11151 using the KpnI and NotI restriction sites and cloned tdTomato into the vector under the CAG promoter using In-Fusion HD Cloning kits (Clonetech Takara Bio, catalog #639649). ..
Clone Assay:Article Title: Impaired AMPARs Translocation into Dendritic Spines with Motor Skill Learning in the Fragile X Mouse Model
Article Snippet: .. For the morphologic marker, we excised DsRed2 from addgene Plasmid #11151 using the KpnI and NotI restriction sites and cloned tdTomato into the vector under the CAG promoter using In-Fusion HD Cloning kits (Clonetech Takara Bio, catalog #639649). ..
Cloning:Article Title: Impaired AMPARs Translocation into Dendritic Spines with Motor Skill Learning in the Fragile X Mouse Model
Article Snippet: .. For the morphologic marker, we excised DsRed2 from addgene Plasmid #11151 using the KpnI and NotI restriction sites and cloned tdTomato into the vector under the CAG promoter using In-Fusion HD Cloning kits (Clonetech Takara Bio, catalog #639649). ..
Retroviral:Article Title: NIX initiates mitochondrial fragmentation via DRP1 to drive epidermal differentiation
Article Snippet: The retroviral plasmid pCLBW-Cox8-GFP-mCh (Addgene plasmid #78520) ( ) was used to express the tandem fluorophores EGFP-mCherry linked to the C terminus of the mitochondrial targeting sequence of Cox8. .. All of the remaining retroviral plasmids were constructed by inserting the following tagged protein sequences into the multi-cloning site of the pLZRS retroviral vector (a gift from Dr. Kathleen Green Lab, Northwestern Univ., Chicago, IL): ( 1 ) Mito-dsRed (a gift from Dr. Thomas Schwarz Lab, Harvard Univ., Boston, MA) links the dsRed2 fluorophore to the C terminus of the Cox8 mitochondrial targeting sequence; ( 2 ) TOM20-mCh (from Addgene plasmid #55146 from Dr. Michael Davidson Lab, Florida State Univ., Tallahassee, FL) links mCherry to the C terminus of full-length human TOM20; ( 3 ) GFP-NIX was made by linking EGFP to the N terminus of the full-length human BNIP3L (NIX) AA 1–219 (from Addgene plasmid #17467) ( ) with an intervening linker sequence of 5′-SDLELKL-3′; ( 4 ) GFP-NIX-WA was made by PCR-based site-directed mutagenesis of GFP-NIX to change tryptophan 36 to an alanine residue (W36A); ( 5 ) GFP-NIX-2GA was made by PCR-based site-directed mutagenesis of GFP-NIX to change glycines 204 and 208 to alanine residues (G204A/G208A); ( 6 ) GFP-Mito was made by linking EGFP to the N terminus of AA 186–219 from full-length human BNIP3L (NIX) with an intervening linker sequence of 5′-SDLELKL-3′; ( 7 ) Tubulin-mCh (a gift from Dr. Kathleen Green Lab, Northwestern Univ., Chicago, IL) links the mCherry fluorophore to the C terminus of mouse beta-tubulin; ( 8 ) SNAP-DRP1-K38A (from Addgene plasmid #45161) ( ) links the SNAPf tag to the N terminus of human DRP1 containing a point mutation of lysine 38 to alanine (K38A); ( 9 ) Mito-SNAP was made from Mito-dsRed by replacing dsRed2 with the SNAPf tag at the C terminus of the Cox8 mitochondrial targeting sequence; and ( 10 ) Halo-FIS1 (from Addgene plasmid #111136) ( ) links the Halo7 tag to the N terminus of human FIS1. .. Bafilomycin A1 (BafA1), 100 μM (Sigma #B1793); carbonyl cyanide 3-chlorophenylhydrazone (CCCP), 10 μM (Sigma #C2759); cobalt chloride (CoCl 2 ), 100 μM (Sigma #15862); deferiprone (DFP), 1 mM (Sigma #Y0001976); dimethyloxalylglycine (DMOG), 1 μM (Sigma #D3695); ethyl(2-(5-nitrothiophene-2-carboxamido)thiophene-3-carbonyl)carbamate (EACC), 10 μM (AOBIOUS #AOB13386); EUK134, 50 μM (Sigma #SML0743); Mdivi-1, 25 μM (Cell Signaling #CAS-338967–87-6); MG132, 10 μM (Sigma #M8699).
Construct:Article Title: NIX initiates mitochondrial fragmentation via DRP1 to drive epidermal differentiation
Article Snippet: The retroviral plasmid pCLBW-Cox8-GFP-mCh (Addgene plasmid #78520) ( ) was used to express the tandem fluorophores EGFP-mCherry linked to the C terminus of the mitochondrial targeting sequence of Cox8. .. All of the remaining retroviral plasmids were constructed by inserting the following tagged protein sequences into the multi-cloning site of the pLZRS retroviral vector (a gift from Dr. Kathleen Green Lab, Northwestern Univ., Chicago, IL): ( 1 ) Mito-dsRed (a gift from Dr. Thomas Schwarz Lab, Harvard Univ., Boston, MA) links the dsRed2 fluorophore to the C terminus of the Cox8 mitochondrial targeting sequence; ( 2 ) TOM20-mCh (from Addgene plasmid #55146 from Dr. Michael Davidson Lab, Florida State Univ., Tallahassee, FL) links mCherry to the C terminus of full-length human TOM20; ( 3 ) GFP-NIX was made by linking EGFP to the N terminus of the full-length human BNIP3L (NIX) AA 1–219 (from Addgene plasmid #17467) ( ) with an intervening linker sequence of 5′-SDLELKL-3′; ( 4 ) GFP-NIX-WA was made by PCR-based site-directed mutagenesis of GFP-NIX to change tryptophan 36 to an alanine residue (W36A); ( 5 ) GFP-NIX-2GA was made by PCR-based site-directed mutagenesis of GFP-NIX to change glycines 204 and 208 to alanine residues (G204A/G208A); ( 6 ) GFP-Mito was made by linking EGFP to the N terminus of AA 186–219 from full-length human BNIP3L (NIX) with an intervening linker sequence of 5′-SDLELKL-3′; ( 7 ) Tubulin-mCh (a gift from Dr. Kathleen Green Lab, Northwestern Univ., Chicago, IL) links the mCherry fluorophore to the C terminus of mouse beta-tubulin; ( 8 ) SNAP-DRP1-K38A (from Addgene plasmid #45161) ( ) links the SNAPf tag to the N terminus of human DRP1 containing a point mutation of lysine 38 to alanine (K38A); ( 9 ) Mito-SNAP was made from Mito-dsRed by replacing dsRed2 with the SNAPf tag at the C terminus of the Cox8 mitochondrial targeting sequence; and ( 10 ) Halo-FIS1 (from Addgene plasmid #111136) ( ) links the Halo7 tag to the N terminus of human FIS1. .. Bafilomycin A1 (BafA1), 100 μM (Sigma #B1793); carbonyl cyanide 3-chlorophenylhydrazone (CCCP), 10 μM (Sigma #C2759); cobalt chloride (CoCl 2 ), 100 μM (Sigma #15862); deferiprone (DFP), 1 mM (Sigma #Y0001976); dimethyloxalylglycine (DMOG), 1 μM (Sigma #D3695); ethyl(2-(5-nitrothiophene-2-carboxamido)thiophene-3-carbonyl)carbamate (EACC), 10 μM (AOBIOUS #AOB13386); EUK134, 50 μM (Sigma #SML0743); Mdivi-1, 25 μM (Cell Signaling #CAS-338967–87-6); MG132, 10 μM (Sigma #M8699).
Sequencing:Article Title: NIX initiates mitochondrial fragmentation via DRP1 to drive epidermal differentiation
Article Snippet: The retroviral plasmid pCLBW-Cox8-GFP-mCh (Addgene plasmid #78520) ( ) was used to express the tandem fluorophores EGFP-mCherry linked to the C terminus of the mitochondrial targeting sequence of Cox8. .. All of the remaining retroviral plasmids were constructed by inserting the following tagged protein sequences into the multi-cloning site of the pLZRS retroviral vector (a gift from Dr. Kathleen Green Lab, Northwestern Univ., Chicago, IL): ( 1 ) Mito-dsRed (a gift from Dr. Thomas Schwarz Lab, Harvard Univ., Boston, MA) links the dsRed2 fluorophore to the C terminus of the Cox8 mitochondrial targeting sequence; ( 2 ) TOM20-mCh (from Addgene plasmid #55146 from Dr. Michael Davidson Lab, Florida State Univ., Tallahassee, FL) links mCherry to the C terminus of full-length human TOM20; ( 3 ) GFP-NIX was made by linking EGFP to the N terminus of the full-length human BNIP3L (NIX) AA 1–219 (from Addgene plasmid #17467) ( ) with an intervening linker sequence of 5′-SDLELKL-3′; ( 4 ) GFP-NIX-WA was made by PCR-based site-directed mutagenesis of GFP-NIX to change tryptophan 36 to an alanine residue (W36A); ( 5 ) GFP-NIX-2GA was made by PCR-based site-directed mutagenesis of GFP-NIX to change glycines 204 and 208 to alanine residues (G204A/G208A); ( 6 ) GFP-Mito was made by linking EGFP to the N terminus of AA 186–219 from full-length human BNIP3L (NIX) with an intervening linker sequence of 5′-SDLELKL-3′; ( 7 ) Tubulin-mCh (a gift from Dr. Kathleen Green Lab, Northwestern Univ., Chicago, IL) links the mCherry fluorophore to the C terminus of mouse beta-tubulin; ( 8 ) SNAP-DRP1-K38A (from Addgene plasmid #45161) ( ) links the SNAPf tag to the N terminus of human DRP1 containing a point mutation of lysine 38 to alanine (K38A); ( 9 ) Mito-SNAP was made from Mito-dsRed by replacing dsRed2 with the SNAPf tag at the C terminus of the Cox8 mitochondrial targeting sequence; and ( 10 ) Halo-FIS1 (from Addgene plasmid #111136) ( ) links the Halo7 tag to the N terminus of human FIS1. .. Bafilomycin A1 (BafA1), 100 μM (Sigma #B1793); carbonyl cyanide 3-chlorophenylhydrazone (CCCP), 10 μM (Sigma #C2759); cobalt chloride (CoCl 2 ), 100 μM (Sigma #15862); deferiprone (DFP), 1 mM (Sigma #Y0001976); dimethyloxalylglycine (DMOG), 1 μM (Sigma #D3695); ethyl(2-(5-nitrothiophene-2-carboxamido)thiophene-3-carbonyl)carbamate (EACC), 10 μM (AOBIOUS #AOB13386); EUK134, 50 μM (Sigma #SML0743); Mdivi-1, 25 μM (Cell Signaling #CAS-338967–87-6); MG132, 10 μM (Sigma #M8699).
Polymerase Chain Reaction:Article Title: NIX initiates mitochondrial fragmentation via DRP1 to drive epidermal differentiation
Article Snippet: The retroviral plasmid pCLBW-Cox8-GFP-mCh (Addgene plasmid #78520) ( ) was used to express the tandem fluorophores EGFP-mCherry linked to the C terminus of the mitochondrial targeting sequence of Cox8. .. All of the remaining retroviral plasmids were constructed by inserting the following tagged protein sequences into the multi-cloning site of the pLZRS retroviral vector (a gift from Dr. Kathleen Green Lab, Northwestern Univ., Chicago, IL): ( 1 ) Mito-dsRed (a gift from Dr. Thomas Schwarz Lab, Harvard Univ., Boston, MA) links the dsRed2 fluorophore to the C terminus of the Cox8 mitochondrial targeting sequence; ( 2 ) TOM20-mCh (from Addgene plasmid #55146 from Dr. Michael Davidson Lab, Florida State Univ., Tallahassee, FL) links mCherry to the C terminus of full-length human TOM20; ( 3 ) GFP-NIX was made by linking EGFP to the N terminus of the full-length human BNIP3L (NIX) AA 1–219 (from Addgene plasmid #17467) ( ) with an intervening linker sequence of 5′-SDLELKL-3′; ( 4 ) GFP-NIX-WA was made by PCR-based site-directed mutagenesis of GFP-NIX to change tryptophan 36 to an alanine residue (W36A); ( 5 ) GFP-NIX-2GA was made by PCR-based site-directed mutagenesis of GFP-NIX to change glycines 204 and 208 to alanine residues (G204A/G208A); ( 6 ) GFP-Mito was made by linking EGFP to the N terminus of AA 186–219 from full-length human BNIP3L (NIX) with an intervening linker sequence of 5′-SDLELKL-3′; ( 7 ) Tubulin-mCh (a gift from Dr. Kathleen Green Lab, Northwestern Univ., Chicago, IL) links the mCherry fluorophore to the C terminus of mouse beta-tubulin; ( 8 ) SNAP-DRP1-K38A (from Addgene plasmid #45161) ( ) links the SNAPf tag to the N terminus of human DRP1 containing a point mutation of lysine 38 to alanine (K38A); ( 9 ) Mito-SNAP was made from Mito-dsRed by replacing dsRed2 with the SNAPf tag at the C terminus of the Cox8 mitochondrial targeting sequence; and ( 10 ) Halo-FIS1 (from Addgene plasmid #111136) ( ) links the Halo7 tag to the N terminus of human FIS1. .. Bafilomycin A1 (BafA1), 100 μM (Sigma #B1793); carbonyl cyanide 3-chlorophenylhydrazone (CCCP), 10 μM (Sigma #C2759); cobalt chloride (CoCl 2 ), 100 μM (Sigma #15862); deferiprone (DFP), 1 mM (Sigma #Y0001976); dimethyloxalylglycine (DMOG), 1 μM (Sigma #D3695); ethyl(2-(5-nitrothiophene-2-carboxamido)thiophene-3-carbonyl)carbamate (EACC), 10 μM (AOBIOUS #AOB13386); EUK134, 50 μM (Sigma #SML0743); Mdivi-1, 25 μM (Cell Signaling #CAS-338967–87-6); MG132, 10 μM (Sigma #M8699).
Mutagenesis:Article Title: NIX initiates mitochondrial fragmentation via DRP1 to drive epidermal differentiation
Article Snippet: The retroviral plasmid pCLBW-Cox8-GFP-mCh (Addgene plasmid #78520) ( ) was used to express the tandem fluorophores EGFP-mCherry linked to the C terminus of the mitochondrial targeting sequence of Cox8. .. All of the remaining retroviral plasmids were constructed by inserting the following tagged protein sequences into the multi-cloning site of the pLZRS retroviral vector (a gift from Dr. Kathleen Green Lab, Northwestern Univ., Chicago, IL): ( 1 ) Mito-dsRed (a gift from Dr. Thomas Schwarz Lab, Harvard Univ., Boston, MA) links the dsRed2 fluorophore to the C terminus of the Cox8 mitochondrial targeting sequence; ( 2 ) TOM20-mCh (from Addgene plasmid #55146 from Dr. Michael Davidson Lab, Florida State Univ., Tallahassee, FL) links mCherry to the C terminus of full-length human TOM20; ( 3 ) GFP-NIX was made by linking EGFP to the N terminus of the full-length human BNIP3L (NIX) AA 1–219 (from Addgene plasmid #17467) ( ) with an intervening linker sequence of 5′-SDLELKL-3′; ( 4 ) GFP-NIX-WA was made by PCR-based site-directed mutagenesis of GFP-NIX to change tryptophan 36 to an alanine residue (W36A); ( 5 ) GFP-NIX-2GA was made by PCR-based site-directed mutagenesis of GFP-NIX to change glycines 204 and 208 to alanine residues (G204A/G208A); ( 6 ) GFP-Mito was made by linking EGFP to the N terminus of AA 186–219 from full-length human BNIP3L (NIX) with an intervening linker sequence of 5′-SDLELKL-3′; ( 7 ) Tubulin-mCh (a gift from Dr. Kathleen Green Lab, Northwestern Univ., Chicago, IL) links the mCherry fluorophore to the C terminus of mouse beta-tubulin; ( 8 ) SNAP-DRP1-K38A (from Addgene plasmid #45161) ( ) links the SNAPf tag to the N terminus of human DRP1 containing a point mutation of lysine 38 to alanine (K38A); ( 9 ) Mito-SNAP was made from Mito-dsRed by replacing dsRed2 with the SNAPf tag at the C terminus of the Cox8 mitochondrial targeting sequence; and ( 10 ) Halo-FIS1 (from Addgene plasmid #111136) ( ) links the Halo7 tag to the N terminus of human FIS1. .. Bafilomycin A1 (BafA1), 100 μM (Sigma #B1793); carbonyl cyanide 3-chlorophenylhydrazone (CCCP), 10 μM (Sigma #C2759); cobalt chloride (CoCl 2 ), 100 μM (Sigma #15862); deferiprone (DFP), 1 mM (Sigma #Y0001976); dimethyloxalylglycine (DMOG), 1 μM (Sigma #D3695); ethyl(2-(5-nitrothiophene-2-carboxamido)thiophene-3-carbonyl)carbamate (EACC), 10 μM (AOBIOUS #AOB13386); EUK134, 50 μM (Sigma #SML0743); Mdivi-1, 25 μM (Cell Signaling #CAS-338967–87-6); MG132, 10 μM (Sigma #M8699).
Residue:Article Title: NIX initiates mitochondrial fragmentation via DRP1 to drive epidermal differentiation
Article Snippet: The retroviral plasmid pCLBW-Cox8-GFP-mCh (Addgene plasmid #78520) ( ) was used to express the tandem fluorophores EGFP-mCherry linked to the C terminus of the mitochondrial targeting sequence of Cox8. .. All of the remaining retroviral plasmids were constructed by inserting the following tagged protein sequences into the multi-cloning site of the pLZRS retroviral vector (a gift from Dr. Kathleen Green Lab, Northwestern Univ., Chicago, IL): ( 1 ) Mito-dsRed (a gift from Dr. Thomas Schwarz Lab, Harvard Univ., Boston, MA) links the dsRed2 fluorophore to the C terminus of the Cox8 mitochondrial targeting sequence; ( 2 ) TOM20-mCh (from Addgene plasmid #55146 from Dr. Michael Davidson Lab, Florida State Univ., Tallahassee, FL) links mCherry to the C terminus of full-length human TOM20; ( 3 ) GFP-NIX was made by linking EGFP to the N terminus of the full-length human BNIP3L (NIX) AA 1–219 (from Addgene plasmid #17467) ( ) with an intervening linker sequence of 5′-SDLELKL-3′; ( 4 ) GFP-NIX-WA was made by PCR-based site-directed mutagenesis of GFP-NIX to change tryptophan 36 to an alanine residue (W36A); ( 5 ) GFP-NIX-2GA was made by PCR-based site-directed mutagenesis of GFP-NIX to change glycines 204 and 208 to alanine residues (G204A/G208A); ( 6 ) GFP-Mito was made by linking EGFP to the N terminus of AA 186–219 from full-length human BNIP3L (NIX) with an intervening linker sequence of 5′-SDLELKL-3′; ( 7 ) Tubulin-mCh (a gift from Dr. Kathleen Green Lab, Northwestern Univ., Chicago, IL) links the mCherry fluorophore to the C terminus of mouse beta-tubulin; ( 8 ) SNAP-DRP1-K38A (from Addgene plasmid #45161) ( ) links the SNAPf tag to the N terminus of human DRP1 containing a point mutation of lysine 38 to alanine (K38A); ( 9 ) Mito-SNAP was made from Mito-dsRed by replacing dsRed2 with the SNAPf tag at the C terminus of the Cox8 mitochondrial targeting sequence; and ( 10 ) Halo-FIS1 (from Addgene plasmid #111136) ( ) links the Halo7 tag to the N terminus of human FIS1. .. Bafilomycin A1 (BafA1), 100 μM (Sigma #B1793); carbonyl cyanide 3-chlorophenylhydrazone (CCCP), 10 μM (Sigma #C2759); cobalt chloride (CoCl 2 ), 100 μM (Sigma #15862); deferiprone (DFP), 1 mM (Sigma #Y0001976); dimethyloxalylglycine (DMOG), 1 μM (Sigma #D3695); ethyl(2-(5-nitrothiophene-2-carboxamido)thiophene-3-carbonyl)carbamate (EACC), 10 μM (AOBIOUS #AOB13386); EUK134, 50 μM (Sigma #SML0743); Mdivi-1, 25 μM (Cell Signaling #CAS-338967–87-6); MG132, 10 μM (Sigma #M8699).
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