Mitochondrial Protein Analyses by Western Blot

All protein samples were lysed by homogenization in RIPA buffer. Samples
were run by electrophoresis on polyacrylamide Tris-glycine SDS gels. The
following antibodies were used in the study: NCLX (1:500, NCKX6 Santa Cruz,
sc-161921); MCU (1:1,000, Sigma-Aldrich, HPA016480); MCUb (1:1,000, Abgent,
AP12355b); MICU1 (1:500, custom generation by Yenzyme); EMRE (1:250, Santa Cruz,
sc-86337); LETM1 (1:1,000, Proteintech, 16024-1-AP); VDAC (1:2,500, Abcam,
ab15895); cyclophilin D (1:5,000, Abcam, ab110324); PDH subunits (1:1,000,
Abcam, ab92696), p-PDH^S293 (1:1,000, Abcam, ab110330) ETC respiratory
chain complexes (1:5,000, OxPhos Cocktail, Abcam, MS604) and Licor IR secondary
antibodies (1:12,000). All blots were imaged on a Licor Odyssey system
(anti-mouse, 926-32210; anti-rabbit, 926-68073; anti-goat, 926-32214). Western
blot details have been previously reported in detail^{13 (link)}. All source gels (that is, full-length
western blots) and band density results are available in Supplementary Fig. 1.

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Luongo T.S., Lambert J.P., Gross P., Nwokedi M., Lombardi A.A., Shanmughapriya S., Carpenter A.C., Kolmetzky D., Gao E., van Berlo J.H., Tsai E.J., Molkentin J.D., Chen X., Madesh M., Houser S.R, & Elrod J.W. (2017). The mitochondrial Na+/Ca2+ exchanger is essential for Ca2+ homeostasis and viability. Nature, 545(7652), 93-97.

Publication 2017

ab15895 ab110324 ab92696 ab110330 MS604

Antibodies Buffer Cyclophilin d Electrophoresis Gels Glycine Goat Mouse Polyacrylamide gels Protein Rabbit Ripa Subunits Tris Vdac 1

Corresponding Organization :

Other organizations : Temple University, Ursinus College, University of Minnesota, Columbia University, Cincinnati Children's Hospital Medical Center, Howard Hughes Medical Institute, University of Cincinnati

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Protein expression levels of NCLX, MCU, MCUb, MICU1, EMRE, LETM1, VDAC, cyclophilin D, PDH subunits, p-PDH^S293, and ETC respiratory chain complexes

control variables

Protein samples were lysed by homogenization in RIPA buffer
Samples were run by electrophoresis on polyacrylamide Tris-glycine SDS gels
Antibody concentrations used: NCLX (1:500), MCU (1:1,000), MCUb (1:1,000), MICU1 (1:500), EMRE (1:250), LETM1 (1:1,000), VDAC (1:2,500), cyclophilin D (1:5,000), PDH subunits (1:1,000), p-PDH^S293 (1:1,000), ETC respiratory chain complexes (1:5,000), Licor IR secondary antibodies (1:12,000)
Imaging done on Licor Odyssey system

controls

Positive controls: None explicitly mentioned
Negative controls: None explicitly mentioned

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