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. 2016 Jan 29;44(4):1813–1832. doi: 10.1093/nar/gkw050

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© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 1. — LACTB2 is a mitochondrial, soluble and monomeric protein. (A) Mitochondria localization. HeLa cells were disrupted and the nuclear, cytoplasmic and mitochondria-containing fractions were separated and analyzed for the localization of LACTB2, using an immunoblotting assay. The nucleus-located protein, CPSF3L, was used as marker for nuclear proteins. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as a cytosolic marker and cytochrome C (Cyt C) as a mitochondrial marker. (B) Immunoblot analysis of mitochondrial extract following Proteinase K accessibility assay. TX-100, Triton X-100. PK, Proteinase K. PNPase is a mitochondrial protein located mainly in the intermembrane space and a small amount in the matrix. HSP60 is a mitochondrial matrix protein. Tom20 is a mitochondrial outer membrane protein. (C) LACTB2 is a soluble protein. Immunoblotting analysis of mitochondrial extract following alkaline sodium carbonate (Na₂CO₃) extraction of soluble and peripheral membrane proteins. T, total extract. S, supernatant. P, pellet. Subunit 5 of the ATP synthase (ATP5) was used as a marker for intrinsic membrane proteins and Cyt C as a marker of soluble proteins. (D) Immunoblot analysis of the recombinant LACTB2 (Rec.) and the native protein in isolated mitochondria (Mit.) showing the same size on SDS-PAGE. Therefore, the mitochondria targeting signal is not cleaved upon entering the mitochondria and remain an integral part of the mature protein. (E) LACTB2 is present as a monomer. Soluble proteins of bovine mitochondrial matrix were extracted from bovine liver as described in the methods section. This extract was fractionated on a size exclusion column superdex 200 and analyzed for LACTB2, using an immunoblotting assay. The following proteins were used as size markers: Tyroglobulin (669 kDa), polynucleotide phosphorylase (PNPase) (232 kDa), BSA (67 kDa), β-lactoglobulin (35 kDa) and ribonuclease A (13 kDa).