Plasmids for BNIP3L Protein Characterization

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 BNIP3L^S212A and BNIP3L^S212D 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.

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da Silva Rosa S.C., Martens M.D., Field J.T., Nguyen L., Kereliuk S.M., Hai Y., Chapman D., Diehl-Jones W., Aliani M., West A.R., Thliveris J., Ghavami S., Rampitsch C., Dolinsky V.W, & Gordon J.W. (2020). BNIP3L/Nix-induced mitochondrial fission, mitophagy, and impaired myocyte glucose uptake are abrogated by PRKA/PKA phosphorylation. Autophagy, 17(9), 2257-2272.

Publication 2020

Q5 site directed mutagenesis kit shBNIP3L pLKO.1-puro mEmerald-Mito-7 mCherry-Mito-7 GW1-Mito-pHRed pcDNA3-FLAG-RHEB

Biosensors Calcium Endoplasmic reticulum Gifts Maob Mito Mitochondrial Oligonucleotides Plasmids Site directed mutagenesis

Corresponding Organization : University of Manitoba

Other organizations : Athabasca University, Unité de Nutrition Humaine, Agriculture and Agri-Food Canada

Top 5 similar protocols

Variable analysis

independent variables

MYC-tagged BNIP3L (Myc-Nix)
BNIP3L-CYB5 (Nix-CytoB5)
BNIP3L-MaoB (Nix-MaoB)
BNIP3L-ActA (Nix-ActA)
BNIP3L-ΔTM (Nix-delta TM)
MYC-tagged BNIP3L^S212A
MYC-tagged BNIP3L^S212D
Sh_BNIP3L

dependent variables

Not explicitly mentioned

control variables

Not explicitly mentioned

positive controls

Mitochondrial (CMV-mitoCAR-GECO1) and endoplasmic reticulum (CMV-ER-LAR-GECO1) targeted calcium biosensors

negative controls

Sh_BNIP3L (17,469; deposited by Wafik El-Deiry)

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